Tumor cell vaccines

ABSTRACT

The present disclosure provides an allogeneic whole cell cancer vaccine platform that includes compositions and methods for treating and preventing cancer. Provided herein are compositions containing a therapeutically effective amount of cells from one or more cancer cell lines, some or all of which are modified to (i) inhibit or reduce expression of one or more immunosuppressive factors by the cells, and/or (ii) express or increase expression of one or more immunostimulatory factors by the cells, and/or (iii) express or increase expression of one or more tumor-associated antigens (TAAs), including TAAs that have been mutated, and which comprise cancer cell lines that natively express a heterogeneity of tumor associated antigens and/or neoantigens. Also provided herein are methods of making the vaccine compositions, methods of preparing, and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/109,757 filed Dec. 2, 2020, which claims priority to U.S. ProvisionalPatent Application No. 62/943,055 filed Dec. 3, 2019. The entirecontents of these applications are incorporated herein by reference intheir entirety.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

The Sequence Listing, which is a part of the present disclosure, issubmitted concurrently with the specification as a text file. The nameof the text file containing the Sequence Listing is“54907C_Seqlisting.txt”, which was created on Jul. 7, 2021 and is343,845 bytes in size. The subject matter of the Sequence Listing isincorporated herein in its entirety by reference.

BACKGROUND

Cancer is a leading cause of death. Recent breakthroughs inimmunotherapy approaches, including checkpoint inhibitors, havesignificantly advanced the treatment of cancer, but these approaches areneither customizable nor broadly applicable across indications or to allpatients within an indication. Furthermore, only a subset of patientsare eligible for and respond to these immunotherapy approaches.Therapeutic cancer vaccines have the potential to generate anti-tumorimmune responses capable of eliciting clinical responses in cancerpatients, but many of these therapies have a single target or areotherwise limited in scope of immunomodulatory targets and/or breadth ofantigen specificity. The development of a therapeutic vaccine customizedfor an indication that targets the heterogeneity of the cells within anindividual tumor remains a challenge.

A vast majority of therapeutic cancer vaccine platforms are inherentlylimited in the number of antigens that can be targeted in a singleformulation. The lack of breadth in these vaccines adversely impactsefficacy and can lead to clinical relapse through a phenomenon calledantigen escape, with the appearance of antigen-negative tumor cells.While these approaches may somewhat reduce tumor burden, they do noteliminate antigen-negative tumor cells or cancer stem cells. Harnessinga patient's own immune system to target a wide breadth of antigens couldreduce tumor burden as well as prevent recurrence through the antigenicheterogeneity of the immune response. Thus, a need exists for improvedwhole cell cancer vaccines. Provided herein are methods and compositionsthat address this need.

SUMMARY

In various embodiments, the present disclosure provides an allogeneicwhole cell cancer vaccine platform that includes compositions andmethods for treating and preventing cancer. The present disclosureprovides compositions and methods that are customizable for thetreatment of various solid tumor indications and target theheterogeneity of the cells within an individual tumor. The compositionsand methods of embodiments of the present disclosure are broadlyapplicable across solid tumor indications and to patients afflicted withsuch indications. In some embodiments, the present disclosure providescompositions of cancer cell lines that (i) are modified as describedherein and (ii) express a sufficient number and amount of tumorassociated antigens (TAAs) such that, when administered to a subjectafflicted with a cancer, cancers, or cancerous tumor(s), a TAA-specificimmune response is generated.

In one embodiment, provided herein is a composition comprising atherapeutically effective amount of at least 1 cancer cell line, whereinthe cell line or a combination of the cell lines comprises cells thatexpress at least 5 tumor associated antigens (TAAs) associated with acancer of a subject intended to receive said composition, and whereinsaid composition is capable of eliciting an immune response specific tothe at least 5 TAAs. In another embodiment, provided herein is acomposition comprising a therapeutically effective amount of at least 1cancer cell line, wherein the cell line or a combination of the celllines comprises cells that express at least 10 tumor associated antigens(TAAs) associated with a cancer of a subject intended to receive saidcomposition, and wherein said composition is capable of eliciting animmune response specific to the at least 10 TAAs. In another embodiment,provided herein is a composition comprising a therapeutically effectiveamount of at least 1 cancer cell line, wherein the cell line or acombination of the cell lines comprises cells that express at least 15tumor associated antigens (TAAs) associated with a cancer of a subjectintended to receive said composition, and wherein said composition iscapable of eliciting an immune response specific to the at least 15TAAs. In another embodiment, provided herein is a composition comprisinga therapeutically effective amount of at least 2 cancer cell lines,wherein each cell line or a combination of the cell lines comprisescells that express at least 5 tumor associated antigens (TAAs)associated with a cancer of a subject intended to receive saidcomposition, and wherein each cell line or the combination of the celllines are modified to express or increase expression of at least 1immunostimulatory factor. In another embodiment, provided herein is acomposition comprising a therapeutically effective amount of at least 2cancer cell lines, wherein each cell line or a combination of the celllines comprises cells that express at least 15 tumor associated antigens(TAAs) associated with a cancer of a subject intended to receive saidcomposition, and wherein each cell line or the combination of the celllines are modified to express or increase expression of at least 2immunostimulatory factor. In still another embodiment, provided hereinis an aforementioned composition wherein said composition is capable ofstimulating a 1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25-fold or higher increase in IFNγ production compared to a compositioncomprising unmodified cancer cell lines.

In another embodiment, provided herein is a composition comprising atherapeutically effective amount of at least 2 cancer cell lines,wherein each cell line or a combination of the cell lines comprisescells that express at least 5 tumor associated antigens (TAAs)associated with a cancer of a subject intended to receive saidcomposition, and wherein each cell line or the combination of the celllines are modified to inhibit or decrease expression of at least 1immunosuppressive factor. In another embodiment, provided herein is acomposition comprising a therapeutically effective amount of at least 2cancer cell lines, wherein each cell line or a combination of the celllines comprises cells that express at least 5 tumor associated antigens(TAAs) associated with a cancer of a subject intended to receive saidcomposition, and wherein each cell line or the combination of the celllines are modified to (i) express or increase expression of at least 1immunostimulatory factor, and (ii) inhibit or decrease expression of atleast 1 immunosuppressive factor. In another embodiment, provided hereinis an aforementioned composition wherein each cell line or thecombination of the cell lines comprises cells that express 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 TAAs associated with the cancer of the subjectintended to receive said composition. In another embodiment, thecomposition comprises 2, 3, 4, 5, or 6 cancer cell lines. In stillanother embodiment, each cell line or a combination of the cell linesare modified to express or increase expression of 1, 2, 3, 4, 5, 6, 7,or 8 immunostimulatory factors. In yet another embodiment, each cellline or a combination of the cell lines are modified to inhibit ordecrease expression of 1, 2, 3, 4, 5, 6, 7, or 8 immunosuppressivefactors.

In still another embodiment of the present disclosure, provided hereinis a composition comprising a therapeutically effective amount of atleast 2 cancer cell lines, wherein each cell line or a combination ofthe cell lines comprises cells that are modified to express or increaseexpression of at least 2 immunostimulatory factors. In anotherembodiment, provided herein is a composition comprising atherapeutically effective amount of at least 2 cancer cell lines,wherein each cell line or a combination of the cell lines comprisescells that are modified to express or increase expression of at least 1immunostimulatory factor, and wherein at least 1 of the cell lines ismodified to knockdown or knockout one or more of CD276, TGFβ1, andTGFβ2. In another embodiment, provided herein is a compositioncomprising a therapeutically effective amount of at least 2 cancer celllines, wherein each cell line or a combination of the cell linescomprises cells that are modified to express or increase expression ofat least 1 immunostimulatory factor, and wherein said at least 1immunostimulatory factor increases dendritic cell maturation. In anotherembodiment, provided herein is a composition comprising atherapeutically effective amount of at least 2 cancer cell lines,wherein each cell line or a combination of the cell lines comprisescells that are modified to express or increase expression of at least 1immunostimulatory factor, and wherein said composition is capable ofstimulating a 1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25-fold or higher increase in IFNγ production compared to a compositioncomprising unmodified cancer cell lines. In another embodiment, providedherein is a composition comprising a therapeutically effective amount ofat least 2 cancer cell lines, wherein each cell line or a combination ofthe cell lines comprises cells that are modified to (i) express orincrease expression of at least 1 immunostimulatory factor, and (ii)inhibit or decrease expression of at least 1 immunosuppressive factor,and wherein said composition is capable of stimulating at least a1.5-fold increase in IFNγ production compared to a compositioncomprising unmodified cancer cell lines. In another embodiment, providedherein is a composition comprising a therapeutically effective amount ofat least 2 cancer cell lines, wherein each cell line or a combination ofthe cell lines comprises cells that are modified to (i) express orincrease expression of at least 2 immunostimulatory factors, and (ii)inhibit or decrease expression of at least 1 immunosuppressive factor,and wherein said composition is capable of stimulating at least a1.5-fold increase in IFNγ production compared to a compositioncomprising unmodified cancer cell lines. In still another embodiment,provided herein is a composition comprising a therapeutically effectiveamount of at least 3 cancer cell lines, wherein each cell line or acombination of the cell lines comprises cells that are modified to (i)express or increase expression of at least 2 immunostimulatory factors,and (ii) inhibit or decrease expression of at least 1 immunosuppressivefactor, and wherein said composition is capable of stimulating at leasta 1.7-fold increase in IFNγ production compared to a compositioncomprising unmodified cancer cell lines. In yet another embodiment,provided herein is a composition comprising a therapeutically effectiveamount of at least 3 cancer cell lines, wherein each cell line or acombination of the cell lines comprises cells that are modified to (i)express or increase expression of at least 2 immunostimulatory factors,and (ii) inhibit or decrease expression of at least 2 immunosuppressivefactors, and wherein said composition is capable of stimulating at leasta 2.0-fold increase in IFNγ production compared to a compositioncomprising unmodified cancer cell lines.

In one embodiment, provided herein is an immunogenic compositioncomprising a therapeutically effective amount of at least 1 cancer cellline, wherein the cell line or a combination of the cell lines comprisescells that are modified to (i) express or increase expression of atleast 1 immunostimulatory factor, and (ii) increase expression of atleast 1 tumor associated antigen (TAA) that is either not expressed orminimally expressed by 1 cell line or the combination of the cell lines.In another embodiment, provided herein is an immunogenic compositioncomprising a therapeutically effective amount of at least 2 cancer celllines, wherein the cell line or a combination of the cell linescomprises cells that are modified to (i) express or increase expressionof at least 2 immunostimulatory factors, and (ii) increase expression ofat least 2 tumor associated antigens (TAAs) that are either notexpressed or minimally expressed by 1 cell line or the combination ofthe cell lines. In another embodiment, provided herein is an immunogeniccomposition comprising a therapeutically effective amount of at least 3cancer cell lines, wherein the cell line or a combination of the celllines comprises cells that are modified to (i) express or increaseexpression of at least 2 immunostimulatory factors, and (ii) increaseexpression of at least 2 tumor associated antigens (TAAs) that areeither not expressed or minimally expressed by 1 cell line or thecombination of the cell lines.

In another embodiment, provided herein is an aforemention immunogeniccomposition wherein each cell line or a combination of the cell linesare modified to (i) express or increase expression of 3, 4, 5, 6, 7, 8,9 or 10 immunostimulatory factors, and/or (iii) increase expression of3, 4, 5, 6, 7, 8, 9 or 10 TAAs that are either not expressed orminimally expressed by 1 cell line or the combination of the cell lines.In another embodiment, provided herein is an aforementioned immunogeniccomposition capable of stimulating at least a 1, 1.3, 1.4, 1.5, 1.6,1.7, or 2-fold increase in IFNγ production compared to a compositioncomprising unmodified cancer cell lines.

In yet another embodiment, provided herein is an immunogenic compositioncomprising a therapeutically effective amount of at least 1 cancer cellline, wherein the cell line or a combination of the cell lines comprisescells that are modified to (i) express or increase expression of atleast 1 immunostimulatory factor, (ii) inhibit or decrease expression ofat least 1 immunosuppressive factor, and (iii) increase expression of atleast 1 tumor associated antigen (TAA) that is either not expressed orminimally expressed by 1 cell line or the combination of the cell lines.In another embodiment, provided herein is an immunogenic compositioncomprising a therapeutically effective amount of at least 2 cancer celllines, wherein each cell line or a combination of the cell linescomprises cells that are modified to (i) express or increase expressionof at least 2 immunostimulatory factors, (ii) inhibit or decreaseexpression of at least 2 immunosuppressive factors, and (iii) increaseexpression of at least 2 tumor associated antigens (TAAs) that areeither not expressed or minimally expressed by 1 cell line or thecombination of the cell lines. In another embodiment, provided herein isan immunogenic composition comprising a therapeutically effective amountof at least 3 cancer cell lines, wherein each cell line or a combinationof the cell lines comprises cells that are modified to (i) express orincrease expression of at least 2 immunostimulatory factors, (ii)inhibit or decrease expression of at least 2 immunosuppressive factors,and (iii) increase expression of at least 1 tumor associated antigen(TAA) that is either not expressed or minimally expressed by 1 cell lineor the combination of the cell lines. In another embodiment, providedherein is an immunogenic composition comprising a therapeuticallyeffective amount of at least 3 cancer cell lines, wherein each cell lineor a combination of the cell lines comprises cells that are modified to(i) express or increase expression of at least 2 immunostimulatoryfactors, (ii) inhibit or decrease expression of at least 2immunosuppressive factors, and (iii) increase expression of at least 2tumor associated antigens (TAAs) that are either not expressed orminimally expressed by 1 cell line or the combination of the cell lines.

In some embodiments, an aforementioned immunogenic composition isprovided wherein the composition comprises 4, 5, or 6 cancer cell lines.In some embodiments, each cell line or a combination of the cell linescomprises cells that are modified to increase expression of at least 3,4, 5, 6, 7, 8, 9, or 10 or more TAAs that are either not expressed orminimally expressed by 1 cell line or the combination of the cell lines.In another embodiment, n each cell line or a combination of the celllines are modified to (i) express or increase expression of 3, 4, 5, 6,7, 8, 9 or 10 immunostimulatory factors, (ii) inhibit or decreaseexpression of 3, 4, 5, 6, 7, 8, 9 or 10 immunosuppressive factors,and/or (iii) increase expression of 3, 4, 5, 6, 7, 8, 9 or 10 TAAs thatare either not expressed or minimally expressed by 1 cell line or thecombination of the cell lines.

In still another embodiment of the present disclosure, provided hereinis animmunogenic composition comprising a therapeutically effectiveamount of at least 3 cancer cell lines, wherein each cell line or acombination of the cell lines comprises cells that are modified to (i)express or increase expression of at least 2 immunostimulatory factors,(ii) inhibit or decrease expression of at least 2 immunosuppressivefactors, and/or (iii) express or increase expression of one or more ofCT83, MSLN, TERT, PSMA, MAGEA1, EGFRvIII, hCMV pp65, TBXT, BORIS, FSHR,MAGEA10, MAGEC2, WT1, FBP, TDGF1, Claudin 18, LYK6K, FAP, PRAME,HPV16/18 E6/E7, or mutated versions thereof. In some embodiments, themutated versions comprise: (i) a modified version selected from thegroup consisting of modTERT, modPSMA, modMAGEA1, modTBXT, modBORIS,modFSHR, modMAGEA10, modMAGEC2, modWT1, modKRAS, modFBP, modTDGF1,modClaudin 18, modLY6K, modFAP, and modPRAME; or (ii) a fusion proteinselected from the group consisting of modCT83-MSLN,modMAGEA1-EGFRvIII-pp65, modTBXT-modBORIS, modFSHR-modMAGEA10,modTBXT-modMAGEC2, modTBXT-modWT1, modTBXT-modWT1-KRAS, modWT1-modFBP,modPSMA-modTDGF1, modWT1-modClaudin 18, modPSMA-modLY6K,modFAP-modClaudin 18, and modPRAME-modTBXT. In still other embodiments,the mutated versions comprise: (i) a modified version selected from thegroup consisting of modMesothelin (SEQ ID NO: 62), modTERT (SEQ ID NO:36), modPSMA (SEQ ID NO: 38), modMAGEA1 (SEQ ID NO: 73), modTBXT (SEQ IDNO: 79), modBORIS(SEQ ID NO: 60), modFSHR (SEQ ID NO: 95), modMAGEA10(SEQ ID NO: 97), modMAGEC2 (SEQ ID NO: 87), modWT1 (SEQ ID NO: 81), KRASG12D (SEQ ID NO: 83) or KRAS G12V (SEQ ID NO:85), modFBP (SEQ ID NO:93), modTDGF1 (SEQ ID NO: 89), modClaudin 18 (SEQ ID NO: 110), modLYK6K(SEQ ID NO: 112), modFAP (SEQ ID NO: 115), and modPRAME (SEQ ID NO:99);or (ii) a fusion protein selected from the group consisting of CT83-MSLN(SEQ ID NO: 22), modMAGEA1-EGFRvIII-pp65 (SEQ ID NO: 40),modTBXT-modBORIS (SEQ ID NO:42), modFSHR-modMAGEA10 (SEQ ID NO: 44),modTBXT-modMAGEC2 (SEQ ID NO: 46), modTBXT-modWT1 (SEQ ID NO: 48),modTBXT-modWT1 (KRAS) (SEQ ID NO: 50), modWT1-modFBP (SEQ ID NO: 52),modPSMA-modTDGF1 (SEQ ID NO: 54), modWT1-modClaudin 18 (SEQ ID NO: 56),modPSMA-modLY6K (SEQ ID NO: 58), and modPRAME-modTBXT (SEQ ID NO: 66).

In still another embodiment of the present disclosure, provided hereinis a composition comprising a therapeutically effective amount of acancer stem cell line, wherein said cancer stem cell line is modified toexpress or increase expression of at least 1 immunostimulatory factor.In another embodiment, provided herein is a composition comprising atherapeutically effective amount of a cancer stem cell line, whereinsaid cancer stem cell line is modified to (i) express or increaseexpression of at least 1 immunostimulatory factor, and (ii) inhibit ordecrease expression of at least 1 immunosuppressive factor. In anotherembodiment, provided herein is a composition comprising atherapeutically effective amount of a cancer stem cell line, whereinsaid cell line is modified to (i) express or increase expression of atleast 1 immunostimulatory factor, and (ii) increase expression of atleast 1 TAA that is either not expressed or minimally expressed by thecancer stem cell line. In some embodiments, the at least 1 TAA isselected from the group consisting of TERT, PSMA, MAGEA1, EGFRvIII, hCMVpp65, TBXT, BORIS, FSHR, MAGEA10, MAGEC2, WT1, KRAS, FBP, TDGF1, Claudin18, LY6K, FAP, PRAME, HPV16/18 E6/E7, and FAP, or mutated versionsthereof.

In still another embodiment of the present disclosure, provided hereinis a composition comprising a therapeutically effective amount of acancer stem cell line, wherein said cancer stem cell line is modified to(i) express or increase expression of at least 1 immunostimulatoryfactor, (ii) inhibit or decrease expression of at least 1immunosuppressive factor, and (iii) increase expression of at least 1tumor associated antigen (TAA) that is either not expressed or minimallyexpressed by the cancer stem cell line. In another embodiment, providedherein is a composition comprising a therapeutically effective amount ofa cancer stem cell line, wherein said cancer stem cell line is modifiedto (i) express or increase expression of at least 2 immunostimulatoryfactors, (ii) inhibit or decrease expression of at least 2immunosuppressive factor, and (iii) increase expression of at least 2tumor associated antigens (TAAs) that are either not expressed orminimally expressed by the cancer stem cell line. In some embodiments,the cancer stem cell line is selected from the group consisting ofJHOM-2B, OVCAR-3, OV56, JHOS-4, JHOC-5, OVCAR-4, JHOS-2, EFO-21,CFPAC-1, Capan-1, Panc 02.13, SUIT-2, Panc 03.27, SK-MEL-28, RVH-421, Hs895.T, Hs 940.T, SK-MEL-1, Hs 936.T, SH-4, COLO 800, UACC-62, NCI-H2066,NCI-H1963, NCI-H209, NCI-H889, COR-L47, NCI-H1092, NCI-H1436, COR-L95,COR-L279, NCI-H1048, NCI-H69, DMS 53, HuH-6, Li7, SNU-182, JHH-7,SK-HEP-1, Hep 3B2.1-7, SNU-1066, SNU-1041, SNU-1076, BICR 18, CAL-33,YD-8, CAL-29, KMBC-2, 253J, 253J-BV, SW780, SW1710, VM-CUB-1, BC-3C,KNS-81, TM-31, NMC-G1, GB-1, SNU-201, DBTRG-05MG, YKG-1, ECC10,RERF-GC-1B, TGBC-11-TKB, SNU-620, GSU, KE-39, HuG1-N, NUGC-4, SNU-16,OCUM-1, C2BBe1, Caco-2, SNU-1033, SW1463, COLO 201, GP2d, LoVo, SW403,CL-14, HCC2157, HCC38, HCC1954, HCC1143, HCC1806, HCC1599, MDA-MB-415,CAL-51, K052, SKNO-1, Kasumi-1, Kasumi-6, MHH-CALL-3, MHH-CALL-2, JVM-2,HNT-34, HOS, OUMS-27, T1-73, Hs 870.T, Hs 706.T, SJSA-1, RD-ES, U2O5,SaOS-2, SK-ES-1, MKN-45, HSC-3, HSC-4, DETROIT 562, and SCC-9.

In still another embodiment of the present disclosure, provided hereinis a composition comprising a therapeutically effective amount of smallcell lung cancer cell line DMS 53, wherein said cell line DMS 53 is (i)modified to knockdown TGFβ2, (ii) knockout CD276, and (iii) upregulateexpression of GM-CSF, membrane bound CD40L, and IL-12. In anotherembodiment of the present disclosure, provided herein is a compositioncomprising a therapeutically effective amount of small cell lung cancercell line DMS 53, wherein said cell line DMS 53 is (i) modified toknockdown TGFβ2, (ii) knockout CD276, and (iii) upregulate expression ofGM-CSF and membrane bound CD40L. In still another embodiment of thepresent disclosure, provided herein is a vaccine composition comprisinga therapeutically effective amount of small cell lung cancer cell lineDMS 53, wherein said composition stimulates an immune response specificto at least 1 tumor associated antigen (TAA) expressed by said cell lineDMS 53. In still another embodiment of the present disclosure, providedherein is a composition comprising a therapeutically effective amount ofat least 2 cancer cell lines, wherein at least 1 of the cell linescomprises cells that are modified to express or increase expression ofat least 1 immunostimulatory factor, and wherein at least 1 of the celllines is small cell lung cancer cell line DMS 53 and comprises cellsthat are modified to express or increase expression of at least 1immunostimulatory factor or inhibit or decrease expression of at least 1immunosuppressive factor. In still another embodiment of the presentdisclosure, provided herein is a composition comprising atherapeutically effective amount of at least 2 cancer cell lines,wherein at least 1 cell line comprises cells that are modified toexpress or increase expression of at least 1 immunostimulatory factor,and wherein 1 cell line is small cell lung cancer DMS 53.

In yet another embodiment of the present disclosure, provided herein isa composition comprising a therapeutically effective amount of smallcell lung cancer cell line DMS 53, wherein said cell line is modified to(i) express or increase expression of at least 1 immunostimulatoryfactor, and (ii) inhibit or decrease expression of at least 1immunosuppressive factor. In still another embodiment of the presentdisclosure, provided herein is a composition comprising atherapeutically effective amount of 3 cancer cell lines, wherein eachcell line comprises cells that are modified to (i) express or increaseexpression of at least 2 immunostimulatory factors, and (ii) inhibit ordecrease expression of at least 1 immunosuppressive factors, and wherein1 of the cell lines is small cell lung cancer cell line DMS 53.

In some embodiments, an aforementioned composition is provided whereinsaid composition is a vaccine composition. In some embodiments, anaforementioned composition is provided wherein said composition iscapable of eliciting an immune response in a subject. In someembodiments, an aforementioned composition is provided wherein saidcomposition comprises 3, 4, 5, 6, 7, 8, 9 or 10 cancer cell lines. Insome embodiments, an aforementioned composition is provided wherein saidcomposition comprises modifications to express or increase expression of2, 3, 4, 5, 6, 7, 8, 9, or 10 immunostimulatory factors. In someembodiments, an aforementioned composition is provided wherein saidcomposition comprises modifications to inhibit or decrease expression of2, 3, 4, 5, 6, 7, 8, 9, or 10 immunosuppressive factors. In someembodiments, an aforementioned composition is provided wherein saidcomposition comprises modifications to express or increase expression of2, 3, 4, 5, 6, 7, 8, 9, or 10 TAAs. In one embodiment, the amino acidsequence of one or more of the TAAs has been modified to include amutation or a neoepitope.

In some embodiments of the present disclosure, an aforementionedcomposition is provided wherein said immune response is an innate immuneresponse, an adaptive immune response, a cellular immune response,and/or a humoral response. In one embodiment the immune response is anadaptive immune response. In some embodiments, the adaptive immuneresponse comprises the production of antigen specific cells selectedfrom the group consisting of CD4⁺ T cells, CH⁺ T cells, gamma-delta Tcells, natural killer T cells, and B cells. In other embodiments of thepresent disclosure, the antigen specific CD4⁺ T cells comprise memorycells, T helper type 1 cells, T helper type 9 cells, T helper type 17cells, T helper type 22 cells, and T follicular helper cells. In someembodiments, the antigen specific CD8⁺ T cells comprise memory cells andcytotoxic T lymphocytes. In other embodiments, the antigen specific Bcells comprise memory cells, immunoglobulin M, immunoglobulin G,immunoglobulin D, immunoglobulin E, and immunoglobulin A. In someembodiments, each cell line or a combination of the cell lines expressat least 10 TAAs. In other embodiments, the TAAs are also expressed in acancer of a subject intended to receive said composition.

In some embodiments, an aforementioned composition is provided whereinthe therapeutically effective amount comprises approximately 8×10⁶ cellsof each cell line. In another embodiment, the therapeutically effectiveamount comprises approximately 1×10⁷ cells of each cell line. In someembodiments, the therapeutically effective amount comprisesapproximately 1.0×10⁶-6.0×10⁷ cells of each cell line. In someembodiments, an aforementioned composition is provided wherein thetherapeutically effective amount comprises approximately an equal numberof cells of each cell line. In some embodiments, an aforementionedcomposition is provided herein the cell lines are geneticallyheterogeneous allogeneic, genetically homogeneous allogeneic,genetically heterogeneous xenogeneic, genetically homogeneousxenogeneic, or a combination of allogeneic and xenogeneic.

Provided herein in various embodiments is an aforementioned compositionwherein the cell lines are from parental cell lines of solid tumorsoriginating from the lung, prostate, testis, breast, colon, bladder,gastrointestinal system, brain, spinal cord, urinary tract, colon,rectum, stomach, head and neck, liver, kidney, central nervous system,endocrine system, mesothelium, ovaries, endometrium, pancreas,esophagus, neuroendocrine system, uterus, or skin. In some embodiments,the parental cell lines comprise cells selected from the groupconsisting of squamous cells, carcinoma cells, adenocarcinoma cells,adenosquamous cells, large cell cells, small cell cells, sarcoma cells,clear cell carcinoma cells, carcinosarcoma cells, mixed mesodermalcells, and teratocarcinoma cells. In some embodiments, the sarcoma cellscomprise osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma,mesothelioma, fibrosarcoma, angiosarcoma, liposarcoma, glioma,gliosarcoma, astrocytoma, myxosarcoma, mesenchymous or mixed mesodermal.In some embodiments, the cell line or cell lines are non-small cell lungcancer cell lines or small cell lung cancer cell lines. In otherembodiments, the cell lines are selected from the group consisting ofNCI-H460, NCIH520, A549, DMS 53, LK-2, and NCI-H23. In some embodiments,the cell line or cell lines are small cell lung cancer cell lines. Inother embodiments, the cell lines are selected from the group consistingof DMS 114, NCI-H196, NCI-H1092, SBC-5, NCI-H510A, NCI-H889, NCI-H1341,NCIH-1876, NCI-H2029, NCI-H841, DMS 53, and NCI-H1694. In otherembodiments, the cell line or cell lines are prostate cancer cell linesor testicular cancer cell lines. In some embodiments, the cell lines areselected from the group consisting of PC3, DU-145, LNCAP, NEC8, andNTERA-2c1-D1. In some embodiments, the cell line or cell lines arecolorectal cancer cell lines. In other embodiments, the cell lines areselected from the group consisting of HCT-15, RKO, HuTu-80, HCT-116, andLS411N. In some embodiments, the cell line or cell lines are breast ortriple negative breast cancer cell lines. In some embodiments, the celllines are selected from the group consisting of Hs 578T, AU565, CAMA-1,MCF-7, and T-47D. In other embodiments, the cell line or cell lines arebladder or urinary tract cancer cell lines. In some embodiments, thecell lines are selected from the group consisting of UM-UC-3, J82,TCCSUP, HT-1376, and SCaBER. In other embodiments, the cell line or celllines are head and neck cancer cell lines. In some embodiments, the celllines are selected from the group consisting of HSC-4, Detroit 562, KON,HO-1-N-1, and OSC-20. In other embodiments, the cell line or cell linesare gastric or stomach cancer cell lines. In some embodiments, the celllines are selected from the group consisting of Fu97, MKN74, MKN45,OCUM-1, and MKN1. In other embodiments, the cell line or cell lines areliver cancer or hepatocellular cancer (HCC) cell lines. In someembodiments, the cell lines are selected from the group consisting ofHep-G2, JHH-2, JHH-4, JHH-5, JHH-6, Li7, HLF, HuH-1, HuH-6, and HuH-7.In some embodiments, the cell line or cell lines are glioblastoma cancercell lines. In some embodiments, the cell lines are selected from thegroup consisting of DBTRG-05MG, LN-229, SF-126, GB-1, and KNS-60. Inother embodiments, the cell line or cell lines are ovarian cancer celllines. In some embodiments, the cell lines are selected from the groupconsisting of TOV-112D, ES-2, TOV-21G, OVTOKO, and MCAS. In someembodiments, the cell line or cell lines are esophageal cancer celllines. In other embodiments, the cell lines are selected from the groupconsisting of TE-10, TE-6, TE-4, EC-GI-10, OE33, TE-9, TT, TE-11, OE19,and OE21. In some embodiments, the cell line or cell lines are kidney orrenal cell carcinoma cancer cell lines. In some embodiments, the celllines are selected from the group consisting of A-498, A-704, 769-P,786-0, ACHN, KMRC-1, KMRC-2, VMRC-RCZ, and VMRC-RCW. In otherembodiments, the cell line or cell lines are pancreatic cancer celllines. In some embodiments, the cell lines are selected from the groupconsisting of PANC-1, KP-3, KP-4, SUIT-2, and PSN11. In someembodiments, the cell line or cell lines are endometrial cancer celllines. In other embodiments, the cell lines are selected from the groupconsisting of SNG-M, HEC-1-B, JHUEM-3, RL95-2, MFE-280, MFE-296, TEN,JHUEM-2, AN3-CA, and Ishikawa. In some embodiments, the cell line orcell lines are skin or melanoma cancer cell lines. In some embodiments,the cell lines are selected from the group consisting of RPMI-7951,MeWo, Hs 688(A).T, COLO 829, C32, A-375, Hs 294T, Hs 695T, Hs 852T, andA2058. In other embodiments, the cell line or cell lines aremesothelioma cancer cell lines. In some embodiments, the cell lines areselected from the group consisting of NCI-H28, MSTO-211H, IST-Mes1,ACC-MESO-1, NCI-H2052, NCI-H2452, MPP 89, and IST-Mes2.

In some embodiments, the present disclosure provides an aforementionedcomposition further comprising a cancer stem cell line. In someembodiments, the present disclosure provides an aforementionedcomposition further comprising cell line DMS 53. In some embodiments,the present disclosure provides an aforementioned composition wherein 1of the cell lines is of a different cancer than at least 1 of the othercell lines. In another embodiment, at least 3 cell lines are each of thesame type of cancer. In some embodiments, at least 3 cell lines are eachof a different cell histology type or molecular subtype. In someembodiments, the present disclosure provides an aforementionedcomposition wherein the cell histology type is selected from the groupconsisting of squamous, carcinoma, adenocarcinoma, large cell, smallcell, and sarcoma.

In some embodiments, the present disclosure provides an aforementionedcomposition wherein the modification to increase expression of the atleast 1 immunostimulatory factor comprises use of a lentiviral vector orvectors encoding the at least 1 immunostimulatory factor. In oneembodiment, the at least 1 immunostimulatory factor is expressed at alevel at least 2.0-fold higher compared to unmodified cell lines. Inanother embodiment, the at least 1 immunostimulatory factor is selectedfrom the group consisting of GM-CSF, membrane bound CD40L, GITR, IL-15,IL-23, and IL-12. In another embodiment, the immunostimulatory factorsare GM-CSF, membrane bound CD40L, and IL-12. In another embodiment, theimmunostimulatory factors are GM-CSF, membrane bound CD40L, and IL-15.In another embodiment, the GM-CSF comprises SEQ ID NO: 8. In anotherembodiment, the membrane bound CD40L comprises SEQ ID NO: 3. In anotherembodiment, the IL-12 comprises SEQ ID NO: 10.

In some embodiments, the present disclosure provides an aforementionedcomposition wherein the modification to inhibit or decrease expressionof the at least 1 immunosuppressive factor comprises a knockout or aknockdown of said at least 1 immunosuppressive factor. In omembodiments, expression of the at least 1 immunosuppressive factor isdecreased by at least approximately 5, 10, 15, 20, 25, or 30%. Inanother embodiment, the modification is a knockdown.

In some embodiments, the present disclosure provides an aforementionedcomposition wherein the modifications to inhibit or decrease expressionof the at least 1 immunosuppressive factor comprise a combination ofknocking down expression of the at least 1 immunosuppressive factor andknocking out expression of a different immunosuppressive factor. In someembodiments, the at least 1 immunosuppressive factor is selected fromthe group consisting of CD276, CD47, CTLA4, HLA-E, HLA-G, IDO1, IL-10,TGFβ1, TGFβ2, and TGFβ3. In another embodiment, the at least 1immunosuppressive factor is selected from the group consisting of CD276,HLA-E, HLA-G, TGFβ1, and TGFβ2. In another embodiment, theimmunosuppressive factors are TGFβ1, TGFβ2, and CD276. In still anotherembodiment, the immunosuppressive factors are TGFβ2 and CD276. In yetanother embodiment of the present disclosure, the immunosuppressivefactors are TGFβ1 and CD276. In some embodiments, the TGFβ1 is knockeddown using short hairpin RNA comprising SEQ ID NO: 25. In otherembodiments, TGFβ2 is knocked down using short hairpin RNA comprisingSEQ ID NO: 24. In still other embodiments, CD276 is knocked out using azinc finger nuclease pair that targets a CD276 genomic DNA sequencecomprising SEQ ID NO: 26.

In some embodiments, the present disclosure provides an aforementionedcomposition wherein the composition comprises cell lines that express aheterogeneity of HLA supertypes, and wherein at least 2 different HLA-Aand at least 2 HLA-B supertypes are represented. In some embodiments,the composition expresses major histocompatibility complex molecules inthe HLA-A24, HLA-A01, HLA-A03, HLA-B07, HLA-B08, HLA-B27, and HLA-B44supertypes. In other embodiments, the composition expresses majorhistocompatibility complex molecules in the HLA-A24, HLA-A03, HLA-A01,HLA-B07, HLA-B27, and HLA-B44 supertypes. In yet other embodiments, thecomposition expresses HLA-A01, HLA-A03, HLA-B07, HLA-B08, and HLA-B44supertypes. In some embodiments, the present disclosure provides anaforementioned composition wherein the cell line(s) is a geneticallyhomogeneous cell line. In some embodiments, the present disclosureprovides an aforementioned composition wherein the cell line(s) is agenetically heterogeneous cell line.

Various methods are contemplated and provided by the present disclosure.In one embodiment, the present disclosure provides a method ofstimulating an immune response in a subject comprising administering tothe subject a therapeutically effective amount of an aforementionedcomposition. In one embodiment, the present disclosure provides a methodof stimulating an immune response specific to at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 or more tumor associated antigens (TAAs) in a subjectcomprising administering to the subject a therapeutically effectiveamount of an aforementioned composition. In some embodiments, providedherein is a method of stimulating an immune response in a subjectcomprising administering to the subject a therapeutically effectiveamount of 2 aforementioned compositions In one embodiment, providedherein is a method of stimulating an immune response in a subjectcomprising administering to the subject a therapeutically effectiveamount of 2 or more compositions described herein, wherein thecompositions comprise different combinations of cell lines. In oneembodiment, provided herein is a method of stimulating an immuneresponse in a subject comprising administering to the subject atherapeutically effective amount of 2 compositions described herein,wherein the compositions each comprise 3 different cell lines. In someembodiments, the immune response comprises increased production ofantigen specific or vaccine specific immunoglobulin G antibodies. Inother embodiments, the immune response comprises increased production ofone or more of IL-1β, IL-6, IL-8, IL-12, IL-17A, IL-20, IL-22, TNFα,IFNγ, CCLS, or CXCL10. In one embodiment, the immune response comprisesincreased production of IFNγ. In some embodiments, the immune responsecomprises increased production of Granzyme A, Granzyme B, Perforin, andCD107a. In other embodiments, the immune response comprises decreasedlevels of regulatory T cells, mononuclear monocyte derived suppressorcells, and polymorphonuclear derived suppressor cells. In still otherembodiments, the immune response comprises decreased levels ofcirculating tumor cells (CTCs), neutrophil to lymphocyte ratio (NLR),and platelet to lymphocyte ratio (PLR). In other embodiments, the immuneresponse comprises changes in immune infiltrate in the tumormicroenvironment.

In one embodiment, provided herein is a method of treating cancer in asubject comprising administering to the subject a therapeuticallyeffective amount of a composition described herein. In one embodiment,provided herein is a method of treating cancer in a subject comprisingadministering to the subject a therapeutically effective amount of 2 ormore compositions described herein, wherein the compositions comprisedifferent combinations of cell lines. In one embodiment, provided hereinis a method of treating cancer in a subject comprising administering tothe subject a therapeutically effective amount of 2 compositionsdescribed herein, wherein the compositions each comprise 3 differentcell lines. In one embodiment, provided herein is a method of treatingcancer in a subject comprising administering to the subject atherapeutically effective amount of a composition described herein, andfurther comprising administering to the subject a therapeuticallyeffective amount of a chemotherapeutic agent. In one embodiment,provided herein is a method of treating cancer in a subject comprisingadministering to the subject a therapeutically effective amount of oneor more compositions described herein, and further comprisingadministering to the subject a therapeutically effective amount ofcyclophosphamide. In some embodiments, the therapeutically effectiveamount of cyclophosphamide comprises 50 mg/day for 1-10 days prior tothe administration of the therapeutically effective amount of thecomposition.

In one embodiment, the present disclosure provides a method of treatingcancer in a subject comprising administering to the subject atherapeutically effective amount of a composition described herein, andfurther comprising administering to the subject a therapeuticallyeffective amount of a checkpoint inhibitor. In another embodiment, thecheckpoint inhibitor is selected from the group consisting of aninhibitor of CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1,B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA,KIR, BTLA, SIGLEC9, and 2B4. In some embodiments, the checkpointinhibitor is selected from the group consisting of pembrolizumab,avelumab, atezolizumab, cetrelimab, dostarlimab, cemiplimab,spartalizumab, camrelizumab, durvalumab, and nivolumab. In otherembodiments, an aforementioned method is provided further comprisingadministering to the subject an isolated tumor associated antigen (TAA).In one embodiment, provided herein is a method of treating cancer in asubject comprising administering to the subject a therapeuticallyeffective amount of a composition described herein, and furthercomprising administering to the subject one or more inhibitors selectedfrom the group consisting of inhibitors of ALK, PARP, VEGFRs, EGFR,FGFR1-3, HIF1α, PDGFR1-2, c-Met, c-KIT, Her2, Her3, AR, PR, RET, EPHB4,STAT3, Ras, HDAC1-11, mTOR, and CXCR4.

In one embodiment, provided herein is a method of treating cancer in asubject comprising administering to the subject a therapeuticallyeffective amount of a composition provided herein, and furthercomprising administering to the subject a therapeutically effectiveamount of radiation therapy. In one embodiment, provided herein is amethod of treating cancer in a subject comprising administering atherapeutically effective amount of a composition described herein, andfurther comprising administering to the patient a cancer treatmentsurgery. In one embodiment, provided herein is a method of concurrentlytreating two or more cancers in a subject comprising administering tothe subject a therapeutically effective amount of a compositiondescribed herein.

In another embodiment, provided herein is a method of preparing avaccine composition described herein, comprising the steps of: (a)selecting one or more cancer cell lines that express at least, 5, 10, 15or 20 or more TAAs; and (b) modifying each of the one or more cancercell lines of (a), wherein the cell line or a combination of the celllines comprises cells that are modified to (i) express or increaseexpression of at least 1 immunostimulatory factor, and/or (ii) increaseexpression of at least 1 TAA that is either not expressed or minimallyexpressed by 1 cell line or the combination of the cell lines. In oneembodiment, the cell line or a combination of the cell lines comprisescells that are additionally modified to inhibit or decrease expressionof at least 1 immunosuppressive factor. In another embodiment, themodifying step comprises introducing one or more vectors into one ormore of the cell lines. In yet another embodiment, the one or morevectors are lentiviral vectors. In still another embodiment, the methodfurther comprises the step of adapting the modified cell lines to axeno-free media. In another embodiment, the method further comprises thestep of irradiating the cell lines. In another embodiment, the methodfurther comprises the step of adapting the cells to a cryopreservationmedia.

In various embodiments, the represent disclosure provides anaforemention method wherein the composition or compositions areadministered to the subject by a route selected from the groupconsisting of parenteral, enteral, oral, intramuscular, intradermal,subcutaneous, intratumoral, intranodal, intranasal, transdermal,inhalation, mucosal, and topical. In one embodiment, the route isintradermal. In some embodiments, the composition or compositions areadministered to an administration site on the subject selected from thegroup consisting of arm or arms, thigh or thighs, and back. In anotherembodiment, the compositions are intradermally administered at differentadministration sites on the subject. In another embodiment, thecomposition is intradermally administered by injection with a syringepositioned at an angle between 5 and 15 degrees from the surface of theadministration site. In some embodiments, a method of treating cancer ina subject is provided comprising administering to the subject atherapeutically effective amount of a first dose and therapeuticallyeffective amounts of subsequent doses of one or more compositionsprovided herein, wherein the one or more compositions are administered1-24 times in year one, 1-16 times in year two, and 1-14 times in yearthree. In another embodiment, the present disclosure provides a methodof stimulating an immune response in a subject comprising administeringto the subject a first dose of a therapeutically effective amount of twocompositions provided herein, wherein the first four doses areadministered every 21 days up to day 63, and then every 42 days forthree additional doses up to day 189. In one embodiment, the methodfurther comprises administering five additional doses at 42-dayintervals up to day 399, and then at least at two 84-day intervalsthereafter.

In another embodiment, the present disclosure provides a method ofstimulating an immune response in a subject comprising administering tothe subject a first dose and subsequent doses of a therapeuticallyeffective amount of two compositions provided herein, wherein the firstfour doses are administered every 14 days up to day 42, and then every42 days for three additional doses up to day 168. In one embodiment, themethod further comprises administering to the subject five additionaldoses at 42-day intervals up to day 378, and then at least at two 84-dayintervals thereafter.

In another embodiment, the present disclosure provides a method oftreating a cancer in a subject comprising administering to the subject atherapeutically effective amount of two compositions, wherein eachcomposition comprises at least 2 cancer cell lines modified to (i)express or increase expression of at least 1 immunostimulatory factor,(ii) inhibit or decrease expression of at least 1 immunosuppressivefactor, and (iii) increase expression of at least 1 tumor associatedantigen (TAA) that is either not expressed or minimally expressed by 1cell line or the combination of the cell lines, wherein one compositionis administered to the upper body of the subject, and the othercomposition is administered to the lower body of the subject. In anotherembodiment, the present disclosure provides a method of treating acancer in a subject comprising administering to the subject a first doseand subsequent doses of a therapeutically effective amount of twocompositions, wherein each composition comprises at least 2 cancer celllines modified to (i) express or increase expression of one or more ofGM-CSF, IL-12, and membrane bound CD40L, (ii) inhibit or decreaseexpression of one or more of TGFβ1, TGFβ2, and CD276, and (iii) increaseexpression of at least 1 TAA that is either not expressed or minimallyexpressed by 1 cell line or the combination of the cell lines, whereinone composition is administered to the upper body of the subject, andthe other composition is administered to the lower body of the subject.In some embodiments, the methods provided herein further comprisesadministering to the subject one or more therapeutic agents ortreatments. In other embodiments, the subject refrains from treatmentwith other vaccines or therapeutic agents. In some embodiments, thetherapeutic agent or treatment is selected from the group consisting ofradiotherapy, chemotherapy, surgery, small molecule inhibitors, andcheckpoint inhibitors. In one embodiment, the therapeutic agent iscyclophosphamide. In other embodiments, the checkpoint inhibitor isselected from the group consisting of an inhibitor of CTLA-4, 4-1BB(CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM,TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, BTLA, SIGLEC9, and 2B4.In some embodiments, the checkpoint inhibitor is pembrolizumab,avelumab, atezolizumab, cetrelimab, dostarlimab, cemiplimab,spartalizumab, camrelizumab, durvalumab, or nivolumab. In someembodiments, the one or more therapeutic agents or treatments areadministered prior to at least 1 administration of said first doseand/or said subsequent doses. In other embodiments, the one or moretherapeutic agents or treatments are administered prior to,concurrently, or subsequent to each administration of said composition.In still other embodiments, a first therapeutic agent is administeredprior to said first dose, and wherein a second therapeutic agent isadministered concurrently with said first dose and said subsequentdoses.

In another embodiment, the present disclosure provides a method ofstimulating an immune response in a subject comprising: a. administeringto the subject a first dose of a therapeutically effective amount of twocompositions provided herein, wherein said two compositions areadministered concurrently at different sites, and administering to thesubject subsequent doses of said two compositions after administeringsaid first dose, wherein said two compositions are administeredconcurrently at different sites; and b. optionally administering to thesubject therapeutically effective doses cyclophosphamide for 1-10 daysprior to administering the first dose of (a), and optionally for 1-10days prior to administering said subsequent doses of (a); c. optionallyadministering to the subject a checkpoint inhibitor either (i)concurrently with each dose of (a), or (ii) every one, two, three, orfour weeks following the first dose of (a). In another embodiment, thepresent disclosure provides a method of treating cancer in a subjectcomprising: a. administering to the subject a first dose of atherapeutically effective amount of two compositions described herein,and administering to the subject subsequent doses of said twocompositions after administering said first dose, wherein said twocompositions are administered concurrently at different sites; b.optionally administering to the subject cyclophosphamide for 1-10 daysprior to administering the first dose of (a), and optionally for 1-10days prior to administering said subsequent doses of (a); c. optionallyadministering to the subject a checkpoint inhibitor either (i)concurrently with each dose of (a), or (ii) every one, two, three, orfour weeks following the first dose of (a). In another embodiment, thepresent disclosure provides a method of treating cancer in a subjectcomprising: a. administering to the subject a first dose of atherapeutically effective amount of two compositions according to anyone of claims 1-138, and administering to the subject subsequent dosesof said two compositions after administering said first dose, whereinsaid two compositions are administered concurrently at different sites,and wherein said subsequent doses are administered at 3, 6, 9, 15, 21,and 27 weeks following administration of said first dose; b.administering to the subject cyclophosphamide daily for 7 days prior toadministering said first dose and said subsequent doses of (a); c.administering to the subject a checkpoint inhibitor at 3, 6, 9, 12, 15,18, 21, 24, and 27 weeks following said first dose of (a). In oneembodiment, cyclophosphamide is administered orally and the checkpointinhibitor is pembrolizumab and is administered intravenously. In anotherembodiment, cyclophosphamide is administered orally at a dosage of 50 mgand the checkpoint inhibitor is pembrolizumab and is administeredintravenously at a dosage of 200 mg.

In another embodiment, the present disclosure provides a method oftreating cancer in a subject comprising: a. administering to the subjecta first dose of a therapeutically effective amount of two compositionsprovided herein, and administering to the subject subsequent doses ofsaid two compositions after administering said first dose, wherein saidtwo compositions are administered concurrently at different sites, andwherein said subsequent doses are administered at 2, 4, 6, 12, 18, and24 weeks following administration of said first dose; b. administeringto the subject cyclophosphamide daily for 7 days prior to administeringsaid first dose and said subsequent doses of (a); and c. administeringto the subject a checkpoint inhibitor at 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, and 30 weeks following said first dose of (a). Inone embodiment, cyclophosphamide is administered orally at a dosage of50 mg and the checkpoint inhibitor is durvalumab and is administeredintravenously at a dosage of 10 mg/kg. In other embodiments, the methodsfurther comprise the step of abstaining from cannabinoid administrationfor 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to administration of thecompositions and 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days afteradministration of the compositions.

In some embodiments, each embraced in groups or individually, thesubject suffers from a cancer selected from the group consisting of lungcancer, prostate cancer, breast cancer, esophageal cancer, colorectalcancer, bladder cancer, gastric cancer, head and neck cancer, livercancer, renal cancer, glioma, endometrial cancer, ovarian cancer,pancreatic cancer, melanoma, and mesothelioma. In one embodiment, thebreast cancer is triple negative breast cancer. In another embodiment,the glioma is an astrocytoma. In still another embodiment, theastrocytoma is glioblastoma multiform (GBM).

The present disclosure also provides kits. In one embodiment, thepresent disclosure provides a kit comprising one or more compositionsprovided herein. In another embodiment, the present disclosure providesa kit comprising at least 1 vial, said vial comprising a compositiondescribed herein. In another embodiment, the present disclosure providesa kit comprising a first vaccine composition in a first vial and asecond vaccine composition in a second vial, wherein said first andsecond vaccine compositions each comprise at least 2 cancer cell linesthat are modified to express or increase expression of at least 2immunostimulatory factors. In yet another embodiment, the presentdisclosure provides a A kit comprising 6 vials, wherein the vials eachcontain a composition comprising a cancer cell line, and wherein atleast 4 of the 6 vials comprise a cancer cell line that is modified to(i) express or increase expression of at least 2 immunostimulatoryfactors, and/or (ii) inhibit or decrease expression of at least 2immunosuppressive factors, and/or (iii) increase expression of at least1 TAA that is either not expressed or minimally expressed by 1 cell lineor the combination of the cell lines, wherein at least 4 of the vialscontain different compositions. In some embodiments, the kit furthercomprises instructions for use. In some embodiments, the kit is used forthe treatment of cancer.

Unit doses of the composition provided herein are also contemplated. Inone embodiment, the present disclosure provides a unit dose of amedicament for treating cancer comprising 6 compositions of differentcancer cell lines, wherein at least 4 compositions comprise a cell linethat is modified to (i) express or increase expression of at least 2immunostimulatory factors, and (ii) inhibit or decrease expression of atleast 2 immunosuppressive factors. In some embodiments, cell linescomprise: (a) non-small cell lung cancer cell lines and/or small celllung cancer cell lines selected from the group consisting of NCI-H460,NCIH520, A549, DMS 53, LK-2, and NCI-H23; (b) DMS 53 and five small celllung cancer cell lines selected from the group consisting of DMS 114,NCI-H196, NCI-H1092, SBC-5, NCI-H510A, NCI-H889, NCI-H1341, NCIH-1876,NCI-H2029, NCI-H841, DMS 53, and NCI-H1694; (c) DMS 53 and prostatecancer cell lines or testicular cancer cell lines PC3, DU-145, LNCAP,NEC8, and NTERA-2c1-D1; (d) DMS 53 and colorectal cancer cell linesHCT-15, RKO, HuTu-80, HCT-116, and LS411N; (e) DMS 53 and breast ortriple negative breast cancer cell lines Hs 578T, AU565, CAMA-1, MCF-7,and T-47D; (f) DMS 53 and bladder or urinary tract cancer cell linesUM-UC-3, J82, TCCSUP, HT-1376, and SCaBER; (g) DMS 53 and head or neckcancer cell lines HSC-4, Detroit 562, KON, HO-1-N-1, and OSC-20; (h) DMS53 and gastric or stomach cancer cell lines Fu97, MKN74, MKN45, OCUM-1,and MKN1; (i) DMS 53 and five liver cancer or hepatocellular cancer(HCC) cell lines selected from the group consisting of Hep-G2, JHH-2,JHH-4, JHH-5, JHH-6, Li7, HLF, HuH-1, HuH-6, and HuH-7; (j) DMS 53 andglioblastoma cancer cell lines DBTRG-05MG, LN-229, SF-126, GB-1, andKNS-60; (k) DMS 53 and ovarian cancer cell lines selected from the groupconsisting of TOV-112D, ES-2, TOV-21G, OVTOKO, and MCAS; (l) DMS 53 andfive esophageal cancer cell lines selected from the group consisting ofTE-10, TE-6, TE-4, EC-GI-10, OE33, TE-9, TT, TE-11, OE19, and OE21; (m)DMS 53 and five kidney or renal cell carcinoma cancer cell linesselected from the group consisting of A-498, A-704, 769-P, 786-0, ACHN,KMRC-1, KMRC-2, VMRC-RCZ, and VMRC-RCW; (n) DMS 53 and pancreatic cancercell lines PANC-1, KP-3, KP-4, SUIT-2, and PSN11; (o) DMS 53 and fiveendometrial cancer cell lines selected from the group consisting ofSNG-M, HEC-1-B, JHUEM-3, RL95-2, MFE-280, MFE-296, TEN, JHUEM-2, AN3-CA,and Ishikawa; (p) DMS 53 and five skin or melanoma cancer cell linesselected from the group consisting of RPMI-7951, MeWo, Hs 688(A).T, COLO829, C32, A-375, Hs 294T, Hs 695T, Hs 852T, and A2058; or (q) DMS 53 andfive mesothelioma cancer cell lines selected from the group consistingof NCI-H28, MSTO-211H, IST-Mes1, ACC-MESO-1, NCI-H2052, NCI-H2452, MPP89, and IST-Mes2.

In another embodiment, the present disclosure provides a unit dose of amedicament for treating cancer comprising 6 compositions of differentcancer cell lines, wherein each cell line is modified to (i) express orincrease expression of at least 2 immunostimulatory factors, (ii)inhibit or decrease expression of at least 2 immunosuppressive factors,and/or (iii) express or increase expression of at least 1 TAA that iseither not expressed or minimally expressed by the cancer cell lines. Insome embodiments, two compositions comprising 3 cell lines each aremixed.

In another embodiment, the present disclosure provides a vaccinecomposition comprising therapeutically effective amounts of lung cancercell lines NCI-H460, NCI-H520, and A549; wherein (a) NCI-H460 ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L, and (ii) decrease expression of TGFβ1, TGFβ2, and CD276; (b)NCI-H520 is modified to (i) increase expression of GM-CSF and membranebound CD40L, and (ii) decrease expression of TGFβ1, TGFβ2, and CD276;and (c) A549 is modified to (i) increase expression of GM-CSF, IL-12,and membrane bound CD40L, and (ii) decrease expression of TGFβ1, TGFβ2,and CD276. In another embodiment, the present disclosure provides avaccine composition comprising therapeutically effective amounts of lungcancer cell lines NCI-H460, NCIH520, and A549; wherein (a) NCI-H460 ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L, and (ii) decrease expression of TGFβ1, TGFβ2, and CD276; (b)NCI-H520 is modified to (i) increase expression of GM-CSF and membranebound CD40L, and (ii) decrease expression of TGFβ1, TGFβ2, and CD276;and (c) A549 is modified to (i) increase expression of GM-CSF, IL-12,and membrane bound CD40L, and (ii) decrease expression of TGFβ1, TGFβ2,and CD276; wherein said therapeutically effective amount isapproximately 1.0×10⁷ cells for each cell line or approximately 6×10⁷cells. In still another embodiment, the present disclosure provides avaccine composition comprising therapeutically effective amounts of lungcancer cell lines DMS 53, LK-2, and NCI-H23, wherein (a) DMS 53 ismodified to (i) increase expression of GM-CSF and membrane bound CD40L,and (ii) decrease expression of TGFβ2 and CD276; (b) LK-2 is modified to(i) increase expression of GM-CSF and membrane bound CD40L, (ii)decrease expression of TGFβ1, TGFβ2, and CD276, and (iii) to expressMSLN and CT83; and (c) NCI-H23 is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L, and (ii) decrease expression ofTGFβ1, TGFβ2, and CD276. In another embodiment, the present disclosureprovides a vaccine composition comprising therapeutically effectiveamounts of lung cancer cell lines DMS 53, LK-2, and NCI-H23; wherein (a)DMS 53 is modified to (i) increase expression of GM-CSF and membranebound CD40L, and (ii) decrease expression of TGFβ2 and CD276; (b) LK-2is modified to (i) increase expression of GM-CSF and membrane boundCD40L, (ii) decrease expression of TGFβ1, TGFβ2, and CD276, and (iii) toexpress MSLN and CT83; and (c) NCI-H23 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L, and (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; wherein said therapeuticallyeffective amount is approximately 1.0×10⁷ cells for each cell line orapproximately 6×10⁷ cells.

In another embodiment, the present disclosure provides a vaccinecomposition comprising therapeutically effective amounts of cancer celllines LN-229, GB-1, and SF-126, wherein: (a) LN-229 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1 and CD276; and (iii) modified to expressmodPSMA; (b) GB-1 is modified to (i) increase expression of GM-CSF andmembrane bound CD40L; and (ii) decrease expression of TGFβ1 and CD276;and (c) SF-126 is modified to (i) increase expression of GM-CSF, IL-12,and membrane bound CD40L; (ii) decrease expression of TGFβ1, TGFβ2, andCD276; and (iii) modified to express modTERT. In another embodiment, thepresent disclosure provides a vaccine composition comprisingtherapeutically effective amounts of cancer cell lines DBTRG-05MG, KNS60, and DMS 53, wherein: (a) DMS 53 is modified to (i) increaseexpression of GM-CSF and membrane bound CD40L; and (ii) decreaseexpression of TGFβ2 and CD276; (b) DBTRG-05MG is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1 and CD276; and (c) KNS 60 is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modified toexpress modMAGEA1, EGFRvIII, and hCMV pp65.

In yet another embodiment, the present disclosure provides a vaccinecomposition comprising therapeutically effective amounts of cancer celllines HCT-15, RKO, and HuTu-80, wherein: (a) HCT-15 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1 and CD276; (b) RKO is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1 and CD276; and (c) HuTu-80 is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modified toexpress modPSMA. In another embodiment, the present disclosure providesa vaccine composition comprising therapeutically effective amounts ofcancer cell lines HCT-116, LS411N and DMS 53, wherein: (a) HCT-116 ismodified to (i) increase expression of GM-CSF and membrane bound CD40L;(ii) decrease expression of TGFβ1 and CD276; and (iii) modified toexpress modTBXT, modWT1, KRAS G12D and KRAS G12V; (b) LS411N is modifiedto (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L;and (ii) decrease expression of TGFβ1 and CD276; and (c) DMS 53 ismodified to (i) increase expression of GM-CSF and membrane bound CD40L;and (ii) decrease expression of TGFβ2 and CD276. In another embodiment,the present disclosure provides a vaccine composition comprisingtherapeutically effective amounts of cancer cell lines PC3, NEC8,NTERA-2c1-D1, wherein: (a) PC3 is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; and (iii) modified to express modTBXT andmodMAGEC2; (b) NEC8 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; and (ii) decrease expression of CD276;and (c) NTERA-2c1-D1 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; and (ii) decrease expression of CD276.In another embodiment, the present disclosure provides a vaccinecomposition comprising therapeutically effective amounts of cancer celllines DU-145, LNCaP, and DMS 53, wherein: (a) DU-145 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of CD276; and (iii) modified to express modPSMA; (b)LNCaP is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; and (ii) decrease expression of CD276; and (c) DMS53 is modified to (i) increase expression of GM-CSF and membrane boundCD40L; and (ii) decrease expression of TGFβ2 and CD276. In anotherembodiment, the present disclosure provides a vaccine compositioncomprising therapeutically effective amounts of cancer cell lines J82,HT-1376, and TCCSUP, wherein: (a) J82 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ2 and CD276; and (iii) modified to express modPSMA;(b) HT-1376 is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2, andCD276; and (c) TCCSUP is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; and (ii) decrease expression of TGFβ1,TGFβ2, and CD276.

In another embodiment, the present disclosure provides a vaccinecomposition comprising therapeutically effective amounts of cancer celllines SCaBER, UM-UC-3 and DMS 53, wherein: (a) SCaBER is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modified toexpress modWT1 and modFOLR1; (b) UM-UC-3 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decreaseexpression of TGFβ1 and CD276; and (c) DMS 53 is modified to (i)increase expression of GM-CSF and membrane bound CD40L; and (ii)decrease expression of TGFβ2 and CD276. In another embodiment, thepresent disclosure provides a vaccine composition comprisingtherapeutically effective amounts of cancer cell lines OVTOKO, MCAS,TOV-112D, wherein: (a) OVTOKO is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression ofTGFβ1 and CD276; (b) MCAS is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; and (iii) modified to express modhTERT; (c)TOV-112D is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; (ii) decrease expression of TGFβ1, TGFβ2, andCD276; and (iii) modified to express modFSHR and modMAGEA10. In anotherembodiment, the present disclosure provides a vaccine compositioncomprising therapeutically effective amounts of cancer cell linesTOV-21G, ES-2 and DMS 53, wherein: (a) TOV-21G is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of CD276; and (iii) modified to express modWT1 andmodFOLR1; (b) ES2 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ1,TGFβ2, and CD276; and (iii) modified to express modBORIS; and (c) DMS 53is modified to (i) increase expression of GM-CSF and membrane boundCD40L; and (ii) decrease expression of TGFβ2 and CD276.

In another embodiment, the present disclosure provides a vaccinecomposition comprising therapeutically effective amounts of cancer celllines HSC-4, HO-1-N-1, and DETROIT 562, wherein: (a) HSC-4 is modifiedto (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L;(ii) decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modifiedto express modPSMA; (b) HO-1-N-1 is modified to (i) increase expressionof GM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; and (iii) modified to express modPRAME andmodTBXT; and (c) DETROIT 562 is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression ofTGFβ1, TGFβ2, and CD276. In another embodiment, the present disclosureprovides a vaccine composition comprising therapeutically effectiveamounts of cancer cell lines KON, OSC-20 and DMS 53, wherein: (a) KON ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; (ii) decrease expression of TGFβ1, TGFβ2, and CD276; and (iii)modified to express HPV16 E6 and E7 and HPV18 E6 and E7; (b) OSC-20 ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; and (ii) decrease expression of TGFβ2 and CD276; and (c) DMS 53is modified to (i) increase expression of GM-CSF and membrane boundCD40L; and (ii) decrease expression of TGFβ2 and CD276. In anotherembodiment, the present disclosure provides a vaccine compositioncomprising therapeutically effective amounts of cancer cell lines MKN-1,MKN-45, and MKN-74, wherein: (a) MKN-1 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (iii) modified to expressmodPSMA and modLYK6; (b) MKN-45 is modified to (i) increase expressionof GM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression ofTGFβ1 and CD276; and (c) MKN-74 is modified to (i) increase expressionof GM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expressionof TGFβ1, and CD276. In another embodiment, the present disclosureprovides a vaccine composition comprising therapeutically effectiveamounts of cancer cell lines OCUM-1, Fu97 and DMS 53, wherein: (a)OCUM-1 is modified to (i) increase expression of GM-CSF and membranebound CD40L; (ii) decrease expression of CD276; (b) Fu97 is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and(ii) decrease expression of TGFβ1 and CD276; and (iii) modified toexpress modWT1 and modCLDN18; and (c) DMS 53 is modified to (i) increaseexpression of GM-CSF and membrane bound CD40L; and (ii) decreaseexpression of TGFβ2 and CD276.

In another embodiment, the present disclosure provides a vaccinecomposition comprising therapeutically effective amounts of cancer celllines CAMA-1, AU565, and HS-578T, wherein: (a) CAMA-1 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ2, and CD276; and (iii) modified to expressmodPSMA; (b) AU565 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ2 andCD276; and (iii) modified to express modTERT; and (c) HS-578T ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; and (ii) decrease expression of TGFβ1, TGFβ2 and CD276. Inanother embodiment, the present disclosure provides a vaccinecomposition comprising therapeutically effective amounts of cancer celllines MCF-7, T47D and DMS 53, wherein: (a) MCF-7 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1, TGFβ2 and CD276; (b) T47D is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and(ii) decrease expression of CD276; and (iii) modified to express modTBXTand modBORIS; and (c) DMS 53 is modified to (i) increase expression ofGM-CSF and membrane bound CD40L; and (ii) decrease expression of TGFβ2and CD276. In another embodiment, the present disclosure provides anaforementioned vaccine composition wherein said therapeuticallyeffective amount is approximately 1.0×10⁷ cells for each cell line orapproximately 6×10⁷ cells.

In one embodiment, the present disclosure provides a compositioncomprising a first cocktail and a second cocktail; wherein said firstcocktail comprises therapeutically effective amounts of at least 2irradiated cancer cell lines modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L, and (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; and wherein said second cocktail comprises cellline DMS 53 modified to (i) increase expression of GM-CSF and membranebound CD40L, and (ii) decrease expression of TGFβ2 and CD276. In oneembodiment, said first cocktail and/or said second cocktail comprisesone or more cell lines modified to express or increase expression ofCT83, MSLN, TERT, PSMA, MAGEA1, EGFRvIII, hCMV pp65, TBXT, BORIS, FSHR,MAGEA10, MAGEC2, WT1, KRAS, FBP, TDGF1, Claudin 18, LYK6K, PRAME,HPV16/18 E6/E7, or mutated versions thereof.

In another embodiment, the present disclosure provides a method ofstimulating an immune response specific to tumor associated antigens(TAAs) associated with non-small cell lung cancer (NSCLC) in a humansubject comprising administering (i) a therapeutically effective amountof a first vaccine composition comprising therapeutically effectiveamounts of lung cancer cell lines NCI-H460, NCI-H520, and A549; wherein(a) NCI-H460 is modified to (i) increase expression of GM-CSF, IL-12,and membrane bound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2,and CD276; (b) NCI-H520 is modified to (i) increase expression of GM-CSFand membrane bound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2,and CD276; and (c) A549 is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; and (ii) a therapeutically effective amount ofa second vaccine composition comprising therapeutically effectiveamounts of lung cancer cell lines DMS 53, LK-2, and NCI-H23; wherein (d)DMS 53 is modified to (i) increase expression of GM-CSF and membranebound CD40L; and (ii) decrease expression of TGFβ2 and CD276; (e) LK-2is modified to (i) increase expression of GM-CSF and membrane boundCD40L; and (ii) decrease expression of TGFβ1, TGFβ2, and CD276; (iii) toexpress MSLN and CT83; and (f) NCI-H23 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; wherein the first vaccinecomposition is administered intradermally in the subject's arm, and thesecond vaccine composition is administered intradermally in thesubject's thigh. In another embodiment, the present disclosure providesa method of treating non-small cell lung cancer (NSCLC) cancer in ahuman subject comprising administering (i) a therapeutically effectiveamount of a first vaccine composition comprising therapeuticallyeffective amounts of lung cancer cell lines NCI-H460, NCI-H520, andA549; wherein (a) NCI-H460 is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; (b) NCI-H520 is modified to (i) increaseexpression of GM-CSF and membrane bound CD40L; and (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (c) A549 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (ii) atherapeutically effective amount of a second vaccine compositioncomprising therapeutically effective amounts of lung cancer cell linesDMS 53, LK-2, and NCI-H23; wherein (d) DMS 53 is modified to (i)increase expression of GM-CSF, and membrane bound CD40L; and (ii)decrease expression of TGFβ2 and CD276; (e) LK-2 is modified to (i)increase expression of GM-CSF and membrane bound CD40L; and (ii)decrease expression of TGFβ1, TGFβ2, and CD276; (iii) to express MSLNand CT83; and (f) NCI-H23 is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; wherein the first vaccine composition isadministered intradermally in the subject's arm, and the second vaccinecomposition is administered intradermally in the subject's thigh.

In another embodiment, the present disclosure provides a method ofstimulating an immune response specific to tumor associated antigens(TAAs) associated with glioblastoma in a human subject comprisingadministering (i) a therapeutically effective amount of a first vaccinecomposition comprising therapeutically effective amounts of cancer celllines LN-229, GB-1, SF-126; wherein: (a) LN-229 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1 and CD276; and (iii) modified to expressmodPSMA; (b) GB-1 is modified to (i) increase expression of GM-CSF andmembrane bound CD40L; and (ii) decrease expression of TGFβ1 and CD276;and (c) SF-126 is modified to (i) increase expression of GM-CSF, IL-12,and membrane bound CD40L; (ii) decrease expression of TGFβ1, TGFβ2, andCD276; and (iii) modified to express modhTERT; and (ii) atherapeutically effective amount of a second vaccine compositioncomprising therapeutically effective amounts of cancer cell linesDBTRG-05MG, KNS 60, and DMS 53; wherein: (d) DMS 53 is modified to (i)increase expression of GM-CSF and membrane bound CD40L; and (ii)decrease expression of TGFβ2 and CD276; (e) DBTRG-05MG is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and(ii) decrease expression of TGFβ1 and CD276; and (f) KNS 60 is modifiedto (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L;(ii) decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modifiedto express modMAGEA1, EGFRvIII, and hCMV pp65; wherein the first vaccinecomposition is administered intradermally in the subject's arm, and thesecond vaccine composition is administered intradermally in thesubject's thigh. In another embodiment, the present disclosure providesa method of treating glioblastoma in a human subject comprisingadministering (i) a therapeutically effective amount of a first vaccinecomposition comprising therapeutically effective amounts of cancer celllines LN-229, GB-1, SF-126; wherein: (a) LN-229 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1 and CD276; and (iii) modified to expressmodPSMA; (b) GB-1 is modified to (i) increase expression of GM-CSF andmembrane bound CD40L; and (ii) decrease expression of TGFβ1 and CD276;and (c) SF-126 is modified to (i) increase expression of GM-CSF, IL-12,and membrane bound CD40L; (ii) decrease expression of TGFβ1, TGFβ2, andCD276; and (iii) modified to express modTERT; and (ii) a therapeuticallyeffective amount of a second vaccine composition comprisingtherapeutically effective amounts of cancer cell lines DBTRG-05MG, KNS60, and DMS 53; wherein: (d) DMS 53 is modified to (i) increaseexpression of GM-CSF and membrane bound CD40L; and (ii) decreaseexpression of TGFβ2 and CD276; (e) DBTRG-05MG is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1 and CD276; and (f) KNS 60 is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modified toexpress modMAGEA1, EGFRvIII, and hCMV pp65; wherein the first vaccinecomposition is administered intradermally in the subject's arm, and thesecond vaccine composition is administered intradermally in thesubject's thigh.

In another embodiment, the present disclosure provides a method ofstimulating an immune response specific to tumor associated antigens(TAAs) associated with colorectal cancer in a human subject comprisingadministering (i) a therapeutically effective amount of a first vaccinecomposition comprising therapeutically effective amounts of cancer celllines HCT-15, RKO, and HuTu-80, wherein: (a) HCT-15 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1 and CD276; (b) RKO is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1 and CD276; and (c) HuTu-80 is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modified toexpress modPSMA; and (ii) a therapeutically effective amount of a secondvaccine composition comprising therapeutically effective amounts ofcancer cell lines HCT-116, LS411N and DMS 53; wherein: (d) HCT-116 ismodified to (i) increase expression of GM-CSF and membrane bound CD40L;(ii) decrease expression of TGFβ1 and CD276; and (iii) modified toexpress modTBXT, modWT1, KRAS G12D and KRAS G12V; (e) LS411N is modifiedto (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L;and (ii) decrease expression of TGFβ1 and CD276; and (f) DMS 53 ismodified to (i) increase expression of GM-CSF and membrane bound CD40L;and (ii) decrease expression of TGFβ2 and CD276; wherein the firstvaccine composition is administered intradermally in the subject's arm,and the second vaccine composition is administered intradermally in thesubject's thigh. In another embodiment, the present disclosure providesa method of treating colorectal cancer in a human subject comprisingadministering (i) a therapeutically effective amount of a first vaccinecomposition comprising therapeutically effective amounts of cancer celllines HCT-15, RKO, and HuTu-80, wherein: (a) HCT-15 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1 and CD276; (b) RKO is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1 and CD276; and (c) HuTu-80 is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modified toexpress modPSMA; and (ii) a therapeutically effective amount of a secondvaccine composition comprising therapeutically effective amounts ofcancer cell lines HCT-116, LS411N and DMS 53; wherein: (d) HCT-116 ismodified to (i) increase expression of GM-CSF and membrane bound CD40L;(ii) decrease expression of TGFβ1 and CD276; and (iii) modified toexpress modTBXT, modWT1, KRAS G12D and KRAS G12V; (e) LS411N is modifiedto (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L;and (ii) decrease expression of TGFβ1 and CD276; and (f) DMS 53 ismodified to (i) increase expression of GM-CSF and membrane bound CD40L;and (ii) decrease expression of TGFβ2 and CD276; wherein the firstvaccine composition is administered intradermally in the subject's arm,and the second vaccine composition is administered intradermally in thesubject's thigh. In another embodiment, the present disclosure providesa method of stimulating an immune response specific to tumor associatedantigens (TAAs) associated with prostate cancer in a human subjectcomprising administering (i) a therapeutically effective amount of afirst vaccine composition comprising therapeutically effective amountsof cancer cell lines PC3, NEC8, NTERA-2c1-D1, wherein: (a) PC3 ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; (ii) decrease expression of TGFβ1, TGFβ2, and CD276; and (iii)modified to express modTBXT and modMAGEC2; (b) NEC8 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of CD276; and (c) NTERA-2c1-D1 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of CD276; and (ii) a therapeutically effectiveamount of a second vaccine composition comprising therapeuticallyeffective amounts of cancer cell lines DU-145, LNCaP, and DMS 53,wherein: (d) DU-145 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; (ii) decrease expression of CD276; and(iii) modified to express modPSMA; (e) LNCaP is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decreaseexpression of CD276; and (f) DMS 53 is modified to (i) increaseexpression of GM-CSF and membrane bound CD40L; and (ii) decreaseexpression of TGFβ2 and CD276; wherein the first vaccine composition isadministered intradermally in the subject's arm, and the second vaccinecomposition is administered intradermally in the subject's thigh.

In another embodiment, the present disclosure provides a method oftreating prostate cancer in a human subject comprising administering (i)a therapeutically effective amount of a first vaccine compositioncomprising therapeutically effective amounts of cancer cell lines PC3,NEC8, NTERA-2c1-D1, wherein: (a) PC3 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (iii) modified to expressmodTBXT and modMAGEC2; (b) NEC8 is modified to (i) increase expressionof GM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expressionof CD276; and (c) NTERA-2c1-D1 is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression ofCD276; and (ii) a therapeutically effective amount of a second vaccinecomposition comprising therapeutically effective amounts of cancer celllines DU-145, LNCaP, and DMS 53, wherein: (d) DU 145 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of CD276; and (iii) modified to express modPSMA; (e)LNCaP is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; and (ii) decrease expression of CD276; and (f) DMS53 is modified to (i) increase expression of GM-CSF and membrane boundCD40L; and (ii) decrease expression of TGFβ2 and CD276; wherein thefirst vaccine composition is administered intradermally in the subject'sarm, and the second vaccine composition is administered intradermally inthe subject's thigh. In another embodiment, the present disclosureprovides a method of stimulating an immune response specific to tumorassociated antigens (TAAs) associated with bladder cancer in a humansubject comprising administering (i) a therapeutically effective amountof a first vaccine composition comprising therapeutically effectiveamounts of cancer cell lines J82, HT-1376, and TCCSUP, wherein: (a) J82is modified to (i) increase expression of GM-CSF, IL-12, and membranebound CD40L; (ii) decrease expression of TGFβ2 and CD276; and (iii)modified to express modPSMA; (b) HT-1376 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (c) TCCSUP is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (ii) atherapeutically effective amount of a second vaccine compositioncomprising therapeutically effective amounts of cancer cell linesSCaBER, UM-UC-3 and DMS 53, wherein: (d) SCaBER is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modified toexpress modWT1 and modFOLR1; (e) UM-UC-3 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decreaseexpression of TGFβ1 and CD276; and (f) DMS 53 is modified to (i)increase expression of GM-CSF and membrane bound CD40L; and (ii)decrease expression of TGFβ2 and CD276; wherein the first vaccinecomposition is administered intradermally in the subject's arm, and thesecond vaccine composition is administered intradermally in thesubject's thigh.

In another embodiment, the present disclosure provides a method oftreating bladder cancer in a human subject comprising administering (i)a therapeutically effective amount of a first vaccine compositioncomprising therapeutically effective amounts of cancer cell lines J82,HT-1376, and TCCSUP, wherein: (a) J82 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ2 and CD276; and (iii) modified to express modPSMA;(b) HT-1376 is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2, andCD276; and (c) TCCSUP is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; and (ii) decrease expression of TGFβ1,TGFβ2, and CD276; and (ii) a therapeutically effective amount of asecond vaccine composition comprising therapeutically effective amountsof cancer cell lines SCaBER, UM-UC-3 and DMS 53, wherein: (d) SCaBER ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; (ii) decrease expression of TGFβ1, TGFβ2, and CD276; and (iii)modified to express modWT1 and modFOLR1; (e) UM-UC-3 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1 and CD276; and (f) DMS 53 is modified to(i) increase expression of GM-CSF and membrane bound CD40L; and (ii)decrease expression of TGFβ2 and CD276; wherein the first vaccinecomposition is administered intradermally in the subject's arm, and thesecond vaccine composition is administered intradermally in thesubject's thigh.

In another embodiment, the present disclosure provides a method ofstimulating an immune response specific to tumor associated antigens(TAAs) associated with ovarian cancer in a human subject comprisingadministering (i) a therapeutically effective amount of a first vaccinecomposition comprising therapeutically effective amounts of cancer celllines OVTOKO, MCAS, TOV-112D, wherein: (a) OVTOKO is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1 and CD276; (b) MCAS is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modified toexpress modhTERT; (c) TOV-112D is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; and (iii) modified to express modFSHR andmodMAGEA10; and (ii) a therapeutically effective amount of a secondvaccine composition comprising therapeutically effective amounts ofcancer cell lines TOV-21G, ES-2 and DMS 53, wherein: (d) TOV-21G ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; (ii) decrease expression of CD276; and (iii) modified to expressmodWT1 and modFOLR1; (e) ES2 is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; and (iii) modified to express modBORIS; and (f)DMS 53 is modified to (i) increase expression of GM-CSF and membranebound CD40L; and (ii) decrease expression of TGFβ2 and CD276; whereinthe first vaccine composition is administered intradermally in thesubject's arm, and the second vaccine composition is administeredintradermally in the subject's thigh. In another embodiment, the presentdisclosure provides a method of treating ovarian cancer in a humansubject comprising administering (i) a therapeutically effective amountof a first vaccine composition comprising therapeutically effectiveamounts of cancer cell lines OVTOKO, MCAS, TOV-112D, wherein: (a) OVTOKOis modified to (i) increase expression of GM-CSF, IL-12, and membranebound CD40L; and (ii) decrease expression of TGFβ1 and CD276; (b) MCASis modified to (i) increase expression of GM-CSF, IL-12, and membranebound CD40L; (ii) decrease expression of TGFβ1, TGFβ2, and CD276; and(iii) modified to express modhTERT; (c) TOV-112D is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modified toexpress modFSHR and modMAGEA10; and (ii) a therapeutically effectiveamount of a second vaccine composition comprising therapeuticallyeffective amounts of cancer cell lines TOV-21G, ES-2 and DMS 53,wherein: (d) TOV-21G is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; (ii) decrease expression of CD276; and(iii) modified to express modWT1 and modFOLR1; (e) ES2 is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modified toexpress modBORIS; and (f) DMS 53 is modified to (i) increase expressionof GM-CSF and membrane bound CD40L; and (ii) decrease expression ofTGFβ2 and CD276; wherein the first vaccine composition is administeredintradermally in the subject's arm, and the second vaccine compositionis administered intradermally in the subject's thigh.

In another embodiment, the present disclosure provides a method ofstimulating an immune response specific to tumor associated antigens(TAAs) associated with head and neck cancer in a human subjectcomprising administering (i) a therapeutically effective amount of afirst vaccine composition comprising therapeutically effective amountsof cancer cell lines HSC-4, HO-1-N-1, DETROIT 562, wherein: (a) HSC-4 ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; (ii) decrease expression of TGFβ1, TGFβ2, and CD276; and (iii)modified to express modPSMA; (b) HO-1-N-1 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (iii) modified to expressmodPRAME and modTBXT; and (c) DETROIT 562 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (ii) a therapeuticallyeffective amount of a second vaccine composition comprisingtherapeutically effective amounts of cancer cell lines KON, OSC-20 andDMS 53, wherein: (d) KON is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; and (iii) modified to express HPV16 E6 and E7and HPV18 E6 and E7; (e) OSC-20 is modified to (i) increase expressionof GM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expressionof TGFβ2 and CD276; and (f) DMS 53 is modified to (i) increaseexpression of GM-CSF and membrane bound CD40L; and (ii) decreaseexpression of TGFβ2 and CD276; wherein the first vaccine composition isadministered intradermally in the subject's arm, and the second vaccinecomposition is administered intradermally in the subject's thigh. Inanother embodiment, the present disclosure provides a method of treatinghead and neck cancer in a human subject comprising administering (i) atherapeutically effective amount of a first vaccine compositioncomprising therapeutically effective amounts of cancer cell lines HSC-4,HO-1-N-1, DETROIT 562, wherein: (a) HSC-4 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (iii) modified to expressmodPSMA; (b) HO-1-N-1 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ1,TGFβ2, and CD276; and (iii) modified to express modPRAME and modTBXT;and (c) DETROIT 562 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; and (ii) decrease expression of TGFβ1,TGFβ2, and CD276; and (ii) a therapeutically effective amount of asecond vaccine composition comprising therapeutically effective amountsof cancer cell lines KON, OSC-20 and DMS 53, wherein: (d) KON ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; (ii) decrease expression of TGFβ1, TGFβ2, and CD276; and (iii)modified to express HPV16 E6 and E7 and HPV18 E6 and E7; (e) OSC-20 ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; and (ii) decrease expression of TGFβ2 and CD276; and (f) DMS 53is modified to (i) increase expression of GM-CSF and membrane boundCD40L; and (ii) decrease expression of TGFβ2 and CD276; wherein thefirst vaccine composition is administered intradermally in the subject'sarm, and the second vaccine composition is administered intradermally inthe subject's thigh.

In another embodiment, the present disclosure provides a method ofstimulating an immune response specific to tumor associated antigens(TAAs) associated with gastric cancer in a human subject comprisingadministering (i) a therapeutically effective amount of a first vaccinecomposition comprising therapeutically effective amounts of cancer celllines MKN-1, MKN-45, and MKN-74; wherein (a) MKN-1 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modified toexpress modPSMA and modLYK6; (b) MKN-45 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ1 and CD276; (c) MKN-74 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decreaseexpression of TGFβ1, and CD276; and (ii) a therapeutically effectiveamount of a second vaccine composition comprising therapeuticallyeffective amounts of cancer cell lines OCUM-1, Fu97 and DMS 53, wherein(d) OCUM-1 is modified to (i) increase expression of GM-CSF and membranebound CD40L; (ii) decrease expression of CD276; (e) Fu97 is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and(ii) decrease expression of TGFβ1 and CD276; and (iii) modified toexpress modWT1 and modCLDN18; and (f) DMS 53 is modified to (i) increaseexpression of GM-CSF and membrane bound CD40L; and (ii) decreaseexpression of TGFβ2 and CD276; wherein the first vaccine composition isadministered intradermally in the subject's arm, and the second vaccinecomposition is administered intradermally in the subject's thigh. Inanother embodiment, the present disclosure provides a method of treatinggastric cancer in a human subject comprising administering (i) atherapeutically effective amount of a first vaccine compositioncomprising therapeutically effective amounts of cancer cell lines MKN-1,MKN-45, and MKN-74; wherein (a) MKN-1 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (iii) modified to expressmodPSMA and modLYK6; (b) MKN-45 is modified to (i) increase expressionof GM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression ofTGFβ1 and CD276; (c) MKN-74 is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression ofTGFβ1, and CD276; and (ii) a therapeutically effective amount of asecond vaccine composition comprising therapeutically effective amountsof cancer cell lines OCUM-1, Fu97 and DMS 53, wherein (d) OCUM-1 ismodified to (i) increase expression of GM-CSF and membrane bound CD40L;(ii) decrease expression of CD276; (e) Fu97 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decreaseexpression of TGFβ1 and CD276; and (iii) modified to express modWT1 andmodCLDN18; and (f) DMS 53 is modified to (i) increase expression ofGM-CSF and membrane bound CD40L; and (ii) decrease expression of TGFβ2and CD276; wherein the first vaccine composition is administeredintradermally in the subject's arm, and the second vaccine compositionis administered intradermally in the subject's thigh.

In another embodiment, the present disclosure provides a method ofstimulating an immune response specific to tumor associated antigens(TAAs) associated with breast cancer in a human subject comprisingadministering (i) a therapeutically effective amount of a first vaccinecomposition comprising therapeutically effective amounts of cancer celllines CAMA-1, AU565, HS-578T, MCF-7, T47D and DMS 53, wherein: (a)CAMA-1 is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; (ii) decrease expression of TGFβ2, and CD276; and(iii) modified to express modPSMA; (b) AU565 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ2 and CD276; and (iii) modified to express modTERT;and (c) HS-578T is modified to (i) increase expression of GM-CSF, IL-12,and membrane bound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2and CD276; and (ii) a therapeutically effective amount of a secondvaccine composition comprising therapeutically effective amounts ofcancer cell lines MCF-7, T47D and DMS 53, wherein: (d) MCF-7 is modifiedto (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L;and (ii) decrease expression of TGFβ1, TGFβ2 and CD276; (e) T47D ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; and (ii) decrease expression of CD276; and (iii) modified toexpress modTBXT and modBORIS; and (f) DMS 53 is modified to (i) increaseexpression of GM-CSF and membrane bound CD40L; and (ii) decreaseexpression of TGFβ2 and CD276; wherein the first vaccine composition isadministered intradermally in the subject's arm, and the second vaccinecomposition is administered intradermally in the subject's thigh. Inanother embodiment, the present disclosure provides a method of treatingbreast cancer in a human subject comprising administering (i) atherapeutically effective amount of a first vaccine compositioncomprising therapeutically effective amounts of cancer cell linesCAMA-1, AU565, and HS-578T, wherein: (a) CAMA-1 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ2, and CD276; and (iii) modified to expressmodPSMA; (b) AU565 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ2 andCD276; and (iii) modified to express modTERT; and (c) HS-578T ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; and (ii) decrease expression of TGFβ1, TGFβ2 and CD276; and (ii)a therapeutically effective amount of a second vaccine compositioncomprising therapeutically effective amounts of cancer cell lines MCF-7,T47D and DMS 53, wherein: (d) MCF-7 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decreaseexpression of TGFβ1, TGFβ2 and CD276; (e) T47D is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of CD276; and (iii) modified to express modTBXT andmodBORIS; and (f) DMS 53 is modified to (i) increase expression ofGM-CSF and membrane bound CD40L; and (ii) decrease expression of TGFβ2and CD276; wherein the first vaccine composition is administeredintradermally in the subject's arm, and the second vaccine compositionis administered intradermally in the subject's thigh.

In another embodiment, the present disclosure provides a method ofstimulating an immune response specific to tumor associated antigens(TAAs) associated with NSCLC in a human subject comprising: a. orallyadministering cyclophosphamide daily for one week at a dose of 50mg/day; b. after said one week in (a), further administering a firstdose of a vaccine comprising a first and second composition, wherein thefirst composition comprises therapeutically effective amounts of lungcancer cell lines NCI-H460, NCI-H520, and A549; wherein (a) NCI-H460 ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; and (ii) decrease expression of TGFβ1, TGFβ2, and CD276; (b)NCI-H520 is modified to (i) increase expression of GM-CSF and membranebound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2, and CD276;and (c) A549 is modified to (i) increase expression of GM-CSF, IL-12,and membrane bound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2,and CD276; and the second composition comprises therapeuticallyeffective amounts of lung cancer cell lines DMS 53, LK-2, and NCI-H23;wherein (d) DMS 53 is modified to (i) increase expression of GM-CSF andmembrane bound CD40L; and (ii) decrease expression of TGFβ2 and CD276;(e) LK-2 is modified to (i) increase expression of GM-CSF and membranebound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2, and CD276;(iii) to express MSLN and CT83; and (f) NCI-H23 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; c. aftersaid one week in (a), further administering via injection a first doseof a composition comprising pembrolizumab at a dosage of 200 mg; d.further administering subsequent doses of the first and secondcompositions at 3, 6, 9, 15, 21, and 27 weeks following administrationof said first dose in (b), and wherein 50 mg of cyclophosphamide isorally administered for 7 days leading up to each subsequent dose; e.further administering intravenously subsequent doses of the compositioncomprising pembrolizumab at 3, 6, 9, 12, 15, 18, 21, 24, and 27 weeksfollowing said first dose in (c) at a dosage of 200 mg; wherein thefirst composition is administered intradermally in the subject's arm,and the second composition is administered intradermally in thesubject's thigh.

In still another embodiment, the present disclosure provides a method ofstimulating an immune response specific to tumor associated antigens(TAAs) associated with a cancer in a human subject comprising: a. orallyadministering cyclophosphamide daily for one week at a dose of 50mg/day; b. after said one week in (a), further administering a firstdose of a vaccine comprising a first and second composition, wherein thefirst composition is a composition provided herein; and the secondcomposition is a different composition provided herein; c. after saidone week in (a), further administering via injection a first dose of acomposition comprising pembrolizumab at a dosage of 200 mg; d. furtheradministering subsequent doses of the first and second compositions at3, 6, 9, 15, 21, and 27 weeks following administration of said firstdose in (b), and wherein 50 mg of cyclophosphamide is orallyadministered for 7 days leading up to each subsequent dose; e. furtheradministering intravenously subsequent doses of the compositioncomprising pembrolizumab at 3, 6, 9, 12, 15, 18, 21, 24, and 27 weeksfollowing said first dose in (c) at a dosage of 200 mg; wherein thefirst composition is administered intradermally in the subject's arm,and the second composition is administered intradermally in thesubject's thigh.

In another embodiment, the present disclosure provides a method ofstimulating an immune response specific to TAAs associated with NSCLC ina human subject comprising: a. orally administering cyclophosphamidedaily for one week at a dose of 50 mg/day; b. after said one week in(a), further administering a first dose of a vaccine comprising a firstand second composition, wherein the first composition comprisestherapeutically effective amounts of lung cancer cell lines NCI-H460,NCI-H520, and A549; wherein (a) NCI-H460 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; (b) NCI-H520 is modified to (i)increase expression of GM-CSF and membrane bound CD40L; and (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (c) A549 is modifiedto (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L;and (ii) decrease expression of TGFβ1, TGFβ2, and CD276; and the secondcomposition comprises therapeutically effective amounts of lung cancercell lines DMS 53, LK-2, and NCI-H23; wherein (d) DMS 53 is modified to(i) increase expression of GM-CSF and membrane bound CD40L; and (ii)decrease expression of TGFβ2 and CD276; (e) LK-2 is modified to (i)increase expression of GM-CSF and membrane bound CD40L; and (ii)decrease expression of TGFβ1, TGFβ2, and CD276; (iii) to express MSLNand CT83; and (f) NCI-H23 is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; c. after said one week in (a),further administering via injection a first dose of a compositioncomprising durvalumab at a dosage of 10 mg/kg; d. further administeringsubsequent doses of the first and second compositions at 2, 4, 10, 16,22, and 28 weeks following administration of said first dose in (b), andwherein 50 mg of cyclophosphamide is orally administered for 7 daysleading up to each subsequent dose; e. further administeringintravenously subsequent doses of the composition comprising durvalumabat 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 weeksfollowing said first dose in (c) at a dosage of 10 mg/kg; wherein thefirst composition is administered intradermally in the subject's arm,and the second composition is administered intradermally in thesubject's thigh.

In another embodiment, the present disclosure provides a method ofstimulating an immune response specific to TAAs associated with NSCLC ina human subject comprising: a. orally administering cyclophosphamidedaily for one week at a dose of 50 mg/day; b. after said one week in(a), further administering a first dose of a vaccine comprising a firstand second composition, wherein the first composition is a compositionprovided herein and the second composition is a different compositionprovided herein; c. after said one week in (a), further administeringvia injection a first dose of a composition comprising durvalumab at adosage of 10 mg/kg; d. further administering subsequent doses of thefirst and second compositions at 2, 4, 10, 16, 22, and 28 weeksfollowing administration of said first dose in (b), and wherein 50 mg ofcyclophosphamide is orally administered for 7 days leading up to eachsubsequent dose; e. further administering intravenously subsequent dosesof the composition comprising durvalumab at 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28 and 30 weeks following said first dose in (c) ata dosage of 10 mg/kg; wherein the first composition is administeredintradermally in the subject's arm, and the second composition isadministered intradermally in the subject's thigh.

In yet another embodiment, the present disclosure provides a kitcomprising six vials, wherein each vial comprises cells of lung cancercell lines NCI-H460, NCIH520, A549, DMS 53, LK-2, and NCI-H23, andwherein: (a) NCI-H460 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; and (ii) decrease expression of TGFβ1,TGFβ2, and CD276; (b) NCI-H520 is modified to (i) increase expression ofGM-CSF and membrane bound CD40L; and (ii) decrease expression of TGFβ1,TGFβ2, and CD276; (c) A549 is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; (d) DMS 53 is modified to (i) increaseexpression of GM-CSF, and membrane bound CD40L; and (ii) decreaseexpression of TGFβ2 and CD276; (e) LK-2 is modified to (i) increaseexpression of GM-CSF and membrane bound CD40L; and (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; (iii) to express MSLN and CT83;and (f) NCI-H23 is modified to (i) increase expression of GM-CSF, IL-12,and membrane bound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2,and CD276. In another embodiment, the present disclosure provides a kitcomprising six vials, wherein each vial comprises cells of cancer celllines LN-229, GB-1, SF-126, DBTRG-05MG, KNS 60, and DMS 53, wherein: (a)LN-229 is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; (ii) decrease expression of TGFβ1 and CD276; and(iii) modified to express modPSMA; (b) GB-1 is modified to (i) increaseexpression of GM-CSF and membrane bound CD40L; and (ii) decreaseexpression of TGFβ1 and CD276; (c) SF-126 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (iii) modified to expressmodTERT; (d) DMS 53 is modified to (i) increase expression of GM-CSF andmembrane bound CD40L; and (ii) decrease expression of TGFβ2 and CD276;(e) DBTRG-05MG is modified to (i) increase expression of GM-CSF, IL-12,and membrane bound CD40L; and (ii) decrease expression of TGFβ1 andCD276; and (f) KNS 60 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ1,TGFβ2, and CD276; and (iii) modified to express modMAGEA1, EGFRvIII, andhCMV pp65. In another embodiment, the present disclosure provides a kitcomprising six vials, wherein each vial comprises cells of cancer celllines HCT-15, RKO, HuTu-80, HCT-116, LS411N and DMS 53, wherein: (a)HCT-15 is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; and (ii) decrease expression of TGFβ1 and CD276;(b) RKO is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; and (ii) decrease expression of TGFβ1 and CD276;(c) HuTu-80 is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; (ii) decrease expression of TGFβ1, TGFβ2, andCD276; and (iii) modified to express modPSMA; (d) HCT-116 is modified to(i) increase expression of GM-CSF and membrane bound CD40L; (ii)decrease expression of TGFβ1 and CD276; and (iii) modified to expressmodTBXT, modWT1, KRAS G12D and KRAS G12V; (e) LS411N is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1 and CD276; and (f) DMS 53 is modified to(i) increase expression of GM-CSF and membrane bound CD40L; and (ii)decrease expression of TGFβ2 and CD276.

In still another embodiment, the present disclosure provides a kitcomprising six vials, wherein each vial comprises cells of cancer celllines PC3, NEC8, NTERA-2c1-D1, DU-145, LNCaP, and DMS 53, wherein: (a)PC3 is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; (ii) decrease expression of TGFβ1, TGFβ2, andCD276; and (iii) modified to express modTBXT and modMAGEC2; (b) NEC8 ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; and (ii) decrease expression of CD276; (c) NTERA-2c1-D1 ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; and (ii) decrease expression of CD276; (d) DU-145 is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of CD276; and (iii) modified to express modPSMA; (e)LNCaP is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; and (ii) decrease expression of CD276; and (f) DMS53 is modified to (i) increase expression of GM-CSF and membrane boundCD40L; and (ii) decrease expression of TGFβ2 and CD276. In anotherembodiment, the present disclosure provides a kit comprising six vials,wherein each vial comprises cells of cancer cell lines J82, HT-1376,TCCSUP, SCaBER, UM-UC-3 and DMS 53, wherein: (a) J82 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ2 and CD276; and (iii) modified to expressmodPSMA; (b) HT-1376 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; and (ii) decrease expression of TGFβ1,TGFβ2, and CD276; (c) TCCSUP is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; (d) SCaBER is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (iii) modified to expressmodWT1 and modFOLR1; (e) UM-UC-3 is modified to (i) increase expressionof GM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expressionof TGFβ1 and CD276; and (f) DMS 53 is modified to (i) increaseexpression of GM-CSF and membrane bound CD40L; and (ii) decreaseexpression of TGFβ2 and CD276.

In another embodiment, the present disclosure provides a kit comprisingsix vials, wherein each vial comprises cells of cancer cell linesOVTOKO, MCAS, TOV-112D, TOV-21G, ES-2 and DMS 53, wherein: (a) OVTOKO ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; and (ii) decrease expression of TGFβ1 and CD276; (b) MCAS ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; (ii) decrease expression of TGFβ1, TGFβ2, and CD276; and (iii)modified to express modhTERT; (c) TOV-112D is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (iii) modified to expressmodFSHR and modMAGEA10; (d) TOV-21G is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of CD276; and (iii) modified to express modWT1 and modFOLR1;(e) ES2 is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; (ii) decrease expression of TGFβ1, TGFβ2, andCD276; and (iii) modified to express modBORIS; and (f) DMS 53 ismodified to (i) increase expression of GM-CSF and membrane bound CD40L;and (ii) decrease expression of TGFβ2 and CD276. In another embodiment,the present disclosure provides a kit comprising six vials, wherein eachvial comprises cells of cancer cell lines HSC-4, HO-1-N-1, DETROIT 562,KON, OSC-20 and DMS 53, wherein: (a) HSC-4 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (iii) modified to expressmodPSMA; (b) HO-1-N-1 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ1,TGFβ2, and CD276; and (iii) modified to express modPRAME and modTBXT;(c) DETROIT 562 is modified to (i) increase expression of GM-CSF, IL-12,and membrane bound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2,and CD276; (d) KON is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ1,TGFβ2, and CD276; and (iii) modified to express HPV16 E6 and E7 andHPV18 E6 and E7; (e) OSC-20 is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression ofTGFβ2 and CD276; and (f) DMS 53 is modified to (i) increase expressionof GM-CSF and membrane bound CD40L; and (ii) decrease expression ofTGFβ2 and CD276.

In yet another embodiment, the present disclosure provides a kitcomprising six vials, wherein each vial comprises approximately cells ofcancer cell lines MKN-1, MKN-45, MKN-74, OCUM-1, Fu97 and DMS 53,wherein: (a) MKN-1 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ1,TGFβ2, and CD276; and (iii) modified to express modPSMA and modLYK6; (b)MKN-45 is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; (ii) decrease expression of TGFβ1 and CD276; (c)MKN-74 is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; and (ii) decrease expression of TGFβ1, and CD276;(d) OCUM-1 is modified to (i) increase expression of GM-CSF and membranebound CD40L; (ii) decrease expression of CD276; (e) Fu97 is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and(ii) decrease expression of TGFβ1 and CD276; and (iii) modified toexpress modWT1 and modCLDN18; and (f) DMS 53 is modified to (i) increaseexpression of GM-CSF and membrane bound CD40L; and (ii) decreaseexpression of TGFβ2 and CD276. In another embodiment, the presentdisclosure provides a kit comprising six vials, wherein each vialcomprises cells of cancer cell lines CAMA-1, AU565, HS-578T, MCF-7, T47Dand DMS 53, wherein: (a) CAMA-1 is modified to (i) increase expressionof GM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression ofTGFβ2, and CD276; and (iii) modified to express modPSMA; (b) AU565 ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; (ii) decrease expression of TGFβ2 and CD276; and (iii) modifiedto express modTERT; and (c) HS-578T is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decreaseexpression of TGFβ1, TGFβ2 and CD276; (d) MCF-7 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1, TGFβ2 and CD276; (e) T47D is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and(ii) decrease expression of CD276; and (iii) modified to express modTBXTand modBORIS; and (f) DMS 53 is modified to (i) increase expression ofGM-CSF and membrane bound CD40L; and (ii) decrease expression of TGFβ2and CD276.

In another embodiment, the present disclosure provides a unit dose of alung cancer vaccine comprising six compositions wherein each compositioncomprises approximately 1.0×10⁷ cells of lung cancer cell linesNCI-H460, NCIH520, A549, DMS 53, LK-2, and NCI-H23; wherein: (a)NCI-H460 is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2, andCD276; (b) NCI-H520 is modified to (i) increase expression of GM-CSF andmembrane bound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2, andCD276; (c) A549 is modified to (i) increase expression of GM-CSF, IL-12,and membrane bound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2,and CD276; (d) DMS 53 is modified to (i) increase expression of GM-CSFand membrane bound CD40L; and (ii) decrease expression of TGFβ2 andCD276; (e) LK-2 is modified to (i) increase expression of GM-CSF andmembrane bound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2, andCD276; (iii) to express MSLN and CT83; and (f) NCI-H23 is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and(ii) decrease expression of TGFβ1, TGFβ2, and CD276. In anotherembodiment, the present disclosure provides a unit dose of a cancervaccine comprising six compositions wherein each composition comprisesapproximately 1.0×10⁷ cells of cancer cell lines LN-229, GB-1, SF-126,DBTRG-05MG, KNS 60, and DMS 53, wherein: (a) LN-229 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1 and CD276; and (iii) modified to expressmodPSMA (b) GB-1 is modified to (i) increase expression of GM-CSF andmembrane bound CD40L; and (ii) decrease expression of TGFβ1 and CD276;(c) SF-126 is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; (ii) decrease expression of TGFβ1, TGFβ2, andCD276; and (iii) modified to express modhTERT; (d) DMS 53 is modified to(i) increase expression of GM-CSF and membrane bound CD40L; and (ii)decrease expression of TGFβ2 and CD276; (e) DBTRG-05MG is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and(ii) decrease expression of TGFβ1 and CD276; and (f) KNS 60 is modifiedto (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L;(ii) decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modifiedto express modMAGEA1, EGFRvIII, and hCMV pp65.

In another embodiment, the present disclosure provides a unit dose of acancer vaccine comprising six compositions wherein each compositioncomprises approximately 1.0×10⁷ cells of cancer cell lines HCT-15, RKO,HuTu-80, HCT-116, LS411N and DMS 53, wherein: (a) HCT-15 is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and(ii) decrease expression of TGFβ1 and CD276; (b) RKO is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of TGFβ1 and CD276; (c) HuTu-80 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modified toexpress modPSMA; (d) HCT-116 is modified to (i) increase expression ofGM-CSF and membrane bound CD40L; (ii) decrease expression of TGFβ1 andCD276; and (iii) modified to express modTBXT, modWT1, KRAS G12D and KRASG12V; (e) LS411N is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; and (ii) decrease expression of TGFβ1and CD276; and (f) DMS 53 is modified to (i) increase expression ofGM-CSF and membrane bound CD40L; and (ii) decrease expression of TGFβ2and CD276. In another embodiment, the present disclosure provides a unitdose of a cancer vaccine comprising six compositions wherein eachcomposition comprises approximately 1.0×10⁷ cells of cancer cell linesPC3, NEC8, NTERA-2c1-D1, DU-145, LNCaP, and DMS 53, wherein: (a) PC3 ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; (ii) decrease expression of TGFβ1, TGFβ2, and CD276; and (iii)modified to express modTBXT and modMAGEC2; (b) NEC8 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of CD276; (c) NTERA-2c1-D1 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii)decrease expression of CD276; (d) DU-145 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of CD276; and (iii) modified to express modPSMA; (e) LNCaP ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; and (ii) decrease expression of CD276; and (f) DMS 53 is modifiedto (i) increase expression of GM-CSF and membrane bound CD40L; and (ii)decrease expression of TGFβ2 and CD276.

In another embodiment, the present disclosure provides a unit dose of acancer vaccine comprising six compositions wherein each compositioncomprises approximately 1.0×10⁷ cells of cancer cell lines J82, HT-1376,TCCSUP, SCaBER, UM-UC-3 and DMS 53, wherein: (a) J82 is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ2 and CD276; and (iii) modified to expressmodPSMA; (b) HT-1376 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; and (ii) decrease expression of TGFβ1,TGFβ2, and CD276; (c) TCCSUP is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; (d) SCaBER is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (iii) modified to expressmodWT1 and modFOLR1; (e) UM-UC-3 is modified to (i) increase expressionof GM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expressionof TGFβ1 and CD276; and (f) DMS 53 is modified to (i) increaseexpression of GM-CSF and membrane bound CD40L; and (ii) decreaseexpression of TGFβ2 and CD276. In another embodiment, the presentdisclosure provides a unit dose of a cancer vaccine comprising sixcompositions wherein each composition comprises approximately 1.0×10⁷cells of cancer cell lines OVTOKO, MCAS, TOV-112D, TOV-21G, ES-2 and DMS53, wherein: (a) OVTOKO is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression ofTGFβ1 and CD276; (b) MCAS is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; and (iii) modified to express modTERT; (c)TOV-112D is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; (ii) decrease expression of TGFβ1, TGFβ2, andCD276; and (iii) modified to express modFSHR and modMAGEA10; (d) TOV-21Gis modified to (i) increase expression of GM-CSF, IL-12, and membranebound CD40L; (ii) decrease expression of CD276; and (iii) modified toexpress modWT1 and modFOLR1; (e) ES2 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (iii) modified to expressmodBORIS; and (f) DMS 53 is modified to (i) increase expression ofGM-CSF and membrane bound CD40L; and (ii) decrease expression of TGFβ2and CD276.

In yet another embodiment, the present disclosure provides a unit doseof a cancer vaccine comprising six compositions wherein each compositioncomprises approximately 1.0×10⁷ cells of cancer cell lines HSC-4,HO-1-N-1, DETROIT 562, KON, OSC-20 and DMS 53, wherein: (a) HSC-4 ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; (ii) decrease expression of TGFβ1, TGFβ2, and CD276; and (iii)modified to express modPSMA; (b) HO-1-N-1 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; and (iii) modified to expressmodPRAME and modTBXT; (c) DETROIT 562 is modified to (i) increaseexpression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decreaseexpression of TGFβ1, TGFβ2, and CD276; (d) KON is modified to (i)increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii)decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) modified toexpress HPV16 E6 and E7 and HPV18 E6 and E7; (e) OSC-20 is modified to(i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and(ii) decrease expression of TGFβ2 and CD276; and (f) DMS 53 is modifiedto (i) increase expression of GM-CSF and membrane bound CD40L; and (ii)decrease expression of TGFβ2 and CD276. In another embodiment, thepresent disclosure provides a unit dose of a cancer vaccine comprisingsix compositions wherein each composition comprises approximately1.0×10⁷ cells of cancer cell lines MKN-1, MKN-45, MKN-74, OCUM-1, Fu97and DMS 53, wherein: (a) MKN-1 is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression ofTGFβ1, TGFβ2, and CD276; and (iii) modified to express modPSMA andmodLYK6; (b) MKN-45 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ1 andCD276; (c) MKN-74 is modified to (i) increase expression of GM-CSF,IL-12, and membrane bound CD40L; and (ii) decrease expression of TGFβ1,and CD276; (d) OCUM-1 is modified to (i) increase expression of GM-CSFand membrane bound CD40L; (ii) decrease expression of CD276; (e) Fu97 ismodified to (i) increase expression of GM-CSF, IL-12, and membrane boundCD40L; and (ii) decrease expression of TGFβ1 and CD276; and (iii)modified to express modWT1 and modCLDN18; and (f) DMS 53 is modified to(i) increase expression of GM-CSF and membrane bound CD40L; and (ii)decrease expression of TGFβ2 and CD276.

In still another embodiment, the present disclosure provides a unit doseof a cancer vaccine comprising six compositions wherein each compositioncomprises approximately 1.0×10⁷ cells of cancer cell lines CAMA-1,AU565, HS-578T, MCF-7, T47D and DMS 53, wherein: (a) CAMA-1 is modifiedto (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L;(ii) decrease expression of TGFβ2, and CD276; and (iii) modified toexpress modPSMA; (b) AU565 is modified to (i) increase expression ofGM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression ofTGFβ2 and CD276; and (iii) modified to express modTERT; and (c) HS-578Tis modified to (i) increase expression of GM-CSF, IL-12, and membranebound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2 and CD276 (d)MCF-7 is modified to (i) increase expression of GM-CSF, IL-12, andmembrane bound CD40L; and (ii) decrease expression of TGFβ1, TGFβ2 andCD276; (e) T47D is modified to (i) increase expression of GM-CSF, IL-12,and membrane bound CD40L; and (ii) decrease expression of CD276; and(iii) modified to express modTBXT and modBORIS; and (f) DMS 53 ismodified to (i) increase expression of GM-CSF and membrane bound CD40L;and (ii) decrease expression of TGFβ2 and CD276.

In some embodiments, an aforementioned composition is provided whereinDMS 53 is further modified to increase expression of IL-12. In someembodiments, the present disclosure provides an aforementioned unit dosewherein DMS 53 is further modified to increase expression of IL-12. Inother embodiments, an aforementioned kit is provided wherein DMS 53 isfurther modified to increase expression of IL-12. In still otherembodiments, the present disclosure provides an aforementioned methodwherein DMS 53 is further modified to increase expression of IL-12.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A and B show reduction of HLA-G mRNA and protein expression incells stably transduced with shRNA knocking down HLA-G in comparison tocontrols.

FIGS. 2 A and B show reduction of HLA-G expression increases IFNγproduction.

FIGS. 3 A-C show reduction of CD47 expression in the A549 (FIG. 3A),NCI-H460 (FIG. 3B), and NCI-H520 (FIG. 3C) cell lines by zinc-fingernuclease (ZFN)-mediated gene editing.

FIGS. 4 A and B show reduction of CD47 in the NCI-H520 cell lineincreases phagocytosis (FIG. 4A) by monocyte-derived dendritic cells andmacrophages and increases IFNγ responses (FIG. 4B) in the ELISpot assay.

FIG. 5 shows ZFN-mediated gene editing of PD-L1 in the NCI-H460 cellline results in a 99% decrease in PD-L1 expression.

FIG. 6 shows ZFN-mediated gene editing of BST2 in the NCI-H2009 cellline results in a 98.5% reduction in BST2 expression.

FIGS. 7 A-C show reduction of TGFβ1 and TGFβ2 in NCI-H460 cell line byshRNA (FIG. 7A), Cas9 (FIG. 7B), and ZFN-mediated (FIG. 7C) geneediting.

FIGS. 8 A and B show shRNA mediated knockdown of TGFβ1 and/or TGFβ2 inthe DMS 53 (FIG. 8A) cell line and NCI-H520 (FIG. 8B) cell line.

FIGS. 9 A-E show the reduction of TGFβ1 and/or TGFβ2 in the NCI-H2023(FIG. 9A), NCI-H23 (FIG. 9B), A549 (FIG. 9C), LK-2 (FIG. 9D), andNCI-H1703 (FIG. 9E) cell lines.

FIGS. 10 A-C show that knockdown of TGFβ1, TGFβ2, or TGFβ1 and TGFβ2 inthe NCI-H460 cell line significantly increases IFNγ responses againstthe parental NCI-H460 cells and the Survivin (BIRC5) antigen.

FIGS. 11 A and B show that loading dendritic cells (DCs) with lysatefrom NCI-H520 TGFβ1 KD cells increases IFNγ responses against parentalNCI-H460 cells upon re-stimulation in the IFNγ ELISpot assay and in themixed lymphocyte co-culture assay.

FIG. 12 shows the IFNγ response comparison between TGFβ1 TGFβ2 knockdownand knockout.

FIG. 13 shows the proteomic comparison between TGFβ1 TGFβ2 knockdown andknockout.

FIGS. 14 A-F show IFNγ responses against unmodified parental cell lineselicited by exemplary combinations of TGFβ1 and/or TGFβ2 modified celllines.

FIGS. 15 A and B show IFNγ responses to cancer antigens elicited byexemplary combinations of TGFβ1 and/or TGFβ2 modified cell lines.

FIGS. 16 A and B show reduction of HLA-E expression in the RERF-LC-Ad1cell line increases cellular immune responses.

FIGS. 17 A and B show reduction of CTLA-4 expression in the NCI-H520cell line increases cellular immune responses.

FIGS. 18 A and B show reduction of CD276 in the A549 cell line increasescellular immune responses.

FIGS. 19 A-D show reduction of CD47 expression and TGFβ1 and TGFβ2secretion in the NCI-H2023 cell line.

FIGS. 20 A-D show reduction of CD47 expression and TGFβ1 and TGFβ2secretion in the NCI-H23 cell line.

FIGS. 21 A-D show reduction of CD47 expression and TGFβ1 and TGFβ2secretion in the A549 cell line.

FIGS. 22 A-D show reduction of CD47 expression and TGFβ1 and TGFβ2secretion in the NCI-H460 cell line.

FIGS. 23 A-C show reduction of CD47 expression and TGFβ1 secretion inthe NCI-H1703 cell line.

FIGS. 24 A-C show reduction of CD47 expression and TGFβ2 secretion inthe LK-2 cell line.

FIGS. 25 A-C show reduction of CD47 expression and TGFβ2 secretion inthe DMS 53 cell line.

FIGS. 26 A-C show reduction of CD47 expression and TGFβ2 secretion inthe NCI-H520 cell line.

FIGS. 27 A-D show reduction of CD276 expression and TGFβ1 and TGFβ2secretion in the NCI-H2023 cell line.

FIGS. 28 A-D show reduction of CD276 expression and TGFβ1 and TGFβ2secretion in the NCI-H23 cell line.

FIGS. 29 A-D show reduction of CD276 expression and TGFβ1 and TGFβ2secretion in the A549 cell line.

FIGS. 30 A-D show reduction of CD276 expression and TGFβ1 and TGFβ2secretion in the NCI-H460 cell line.

FIGS. 31 A-C show reduction of CD276 expression and TGFβ1 secretion inthe NCI-H1703 cell line.

FIGS. 32 A-C show reduction of CD276 expression and TGFβ2 secretion inthe LK-2 cell line.

FIGS. 33 A-C show reduction of CD276 expression an TGFβ2 secretion inthe DMS 53 cell line.

FIGS. 34 A-C show reduction of CD276 expression and TGFβ2 secretion inthe NCI-H520 cell line.

FIGS. 35 A and B show reduction of CD276 expression and TGFβ1 and TGFβ2secretion in the NCI-H460 (FIG. 35A) and A549 (FIG. 35B) cell linesincreases cellular immune responses.

FIGS. 36 A-D show reduction of CD47 and CD276 expression and TGFβ1 andTGFβ2 secretion in the A549 cell line.

FIGS. 37 A and B show reduction of CD47 and CD276 expression and TGFβ1and TGFβ2 secretion increases immunogenicity.

FIGS. 38 A-D show expression of membrane bound CD40L in the A549 cellline increases dendritic cell (DC) maturation and cellular immuneresponses.

FIG. 39 shows overexpression of GM-CSF in the NCI-H460 cell lineincreases cellular immune responses.

FIG. 40 shows expression of IL-12 in the A549 cell line increasescellular immune responses.

FIGS. 41 A-D show expression of GITR in the NCI-H520 (FIG. 41A), A549(FIG. 41B), LK-2 (FIG. 41C), and NCI-H460 (FIG. 41D) cell lines.

FIGS. 42 A-D show expression of GITR enhances cellular immune responses.

FIGS. 43 A and B show expression of IL-15 enhances cellular immuneresponses.

FIGS. 44 A and B show expression of IL-23 enhances cellular immuneresponses.

FIG. 45 shows the expression of XCL1.

FIGS. 46 A-E show expression of Mesothelin and increasedmesothelin-specific IFNγ responses in the NCI-H520 cell line (FIG. 46A),LK-2 cell line (FIG. 46B and FIG. 46E), A549 cell line (FIG. 46C), andNCI-H460 cell line (FIG. 46 D).

FIG. 47 shows the expression of CT83.

FIGS. 48 A-E show secretion of GM-CSF and expression of membrane boundCD40L in the A549 TGFβ1 TGFβ2 KD CD47 KO cell line.

FIGS. 49 A-E show secretion of GM-CSF and expression of membrane boundCD40L in the NCI-H460 TGFβ1 TGFβ2 KD CD47 KO cell line.

FIGS. 50 A-E show secretion of GM-CSF and expression of membrane boundCD40L in the A549 TGFβ1 TGFβ2 KD CD276 KO cell line.

FIGS. 51 A-E show secretion of GM-CSF and expression of membrane boundCD40L in the NCI-H460 TGFβ1 TGFβ2 KD CD276 KO cell line.

FIGS. 52 A-C show secretion of GM-CSF and expression of membrane boundCD40L in TGFβ1 TGFβ2 KD CD47 KO or TGFβ1 TGFβ2 KD CD276 KO cell linesincreases cellular immune responses and DC maturation.

FIGS. 53 A-F show secretion of GM-CSF, expression of membrane boundCD40L, and secretion of IL-12 in the A549 TGFβ1 TGFβ2 KD CD47 KO cellline.

FIGS. 54 A-F show secretion of GM-CSF, expression of membrane boundCD40L, and secretion of IL-12 in the NCI-H460 TGFβ1 TGFβ2 KD CD47 KOcell line.

FIGS. 55 A and B show secretion of GM-CSF, expression of membrane boundCD40L, and secretion of IL-12 by the A549 (FIG. 55A) and NCI-H460 (FIG.55B) TGFβ1 TGFβ2 KD CD47 KO cell lines increases antigen specificresponses.

FIG. 56 shows the secretion of GM-CSF, expression of membrane boundCD40L, and secretion of IL-12 in the A549 TGFβ1 TGFβ2 KD CD276 KO cellline.

FIGS. 57 A-F show secretion of GM-CSF, expression of membrane boundCD40L, and secretion of IL-12 in the NCI-H460 TGFβ1 TGFβ2 KD CD276 KOcell line.

FIGS. 58 A-D show secretion of GM-CSF, expression of membrane boundCD40L, and secretion of IL-12 by the A549 and NCI-H460 TGFβ1 TGFβ2 KDCD276 KO cell lines increases DC maturation and antigen specificresponses.

FIG. 59 shows that HLA mismatch results in increased immunogenicity.

FIG. 60 shows the expression of NSCLC antigens in certain cell lines.

FIGS. 61 A-C show a comparison of endogenous TAA expression profiles ofNSCLC vaccines and Belagenpumatucel-L.

FIGS. 62 A and B show IFNγ responses elicited by single lines comparedto cocktails of cell lines.

FIG. 63 shows IFNγ responses against selected antigens.

FIG. 64 shows expression of membrane bound CD40L on the NSCLC vaccinecell lines.

FIGS. 65 A and B show expression of CT83 and Mesothelin by the LK-2 cellline and IFNγ responses to the CT83 and mesothelin antigens.

FIGS. 66 A and B show a comparison of IFNγ responses generated bybelagenpumatucel-L and NSCLC vaccine.

FIGS. 67 A and B show a comparison of IFNγ responses generated bybelagenpumatucel-L and NSCLC vaccine in individual donors.

FIGS. 68 A-C show endogenous expression of GBM antigens (FIG. 68A) andGBM CSC-like markers in candidate vaccine cell lines (FIG. 68B) and GBMpatient tumor samples (FIG. 68C).

FIGS. 69 A-C show IFNγ responses elicited by single candidate GBMvaccine cell lines (FIG. 69A) and in cocktails of cell lines (FIGS.69B-C).

FIGS. 70 A and B show endogenous expression of GBM antigens by the GBMvaccine cell lines (FIG. 70A) and the number of GBM antigens expressedby the vaccine cell lines also expressed in GBM patient tumors (FIG.70B).

FIGS. 71 A-K show the expression of and IFNγ responses to antigensintroduced in the GBM vaccine cell lines compared to unmodifiedcontrols. Expression of modTERT by SF-126 (FIG. 71A) and IFNγ responsesto TERT (FIG. 71G) in GBM-vaccine A. Expression of modPSMA by LN-229(FIG. 71B) and IFNγ responses to PSMA (FIG. 71H) in GBM-vaccine A.Expression of modMAGEA1, EGFRvIII and pp65 by KNS 60 (FIGS. 71C-F) andIFNγ responses to MAGEA1, EGFRvIII and pp65 (FIGS. 71I-K) in GBM-vaccineB.

FIG. 72 shows expression of membrane bound CD40L by the GBM vaccinecomponent cell lines.

FIG. 73 A-C shows antigen specific IFNγ responses induced by the unitdose of the GBM vaccine (FIG. 73A), GBM vaccine-A (FIG. 73B), and GBMvaccine-B (FIG. 73C) compared to unmodified controls.

FIG. 74 shows antigen specific IFNγ responses induced by the unit doseof the GBM vaccine in individual donors compared to unmodified controls.

FIGS. 75 A-C show endogenous expression of CRC antigens (FIG. 75A) andCRC CSC-like markers in selected cell lines (FIG. 75B) and CRC patienttumor samples (FIG. 75C).

FIGS. 76 A-C show IFNγ responses elicited by single candidate CRCvaccine cell lines (FIG. 76A) and in cocktails (FIGS. 76B and C).

FIG. 77 shows IFNγ responses elicited by single candidate CRC vaccinecell lines alone compared to cocktails of cell lines.

FIGS. 78 A and B shows endogenous expression of CRC antigens by the CRCvaccine cell lines (FIG. 78A) and the number of CRC antigens expressedby the vaccine cell lines also expressed in CRC patient tumors (FIG.78B).

FIGS. 79 A-J show the expression of and IFNγ responses to antigensintroduced in the CRC vaccine cell lines compared to unmodifiedcontrols. Expression of modPSMA by HuTu80 (FIG. 79A) and IFNγ responsesto PSMA (FIG. 79F) in CRC-vaccine A. Expression of modTBXT, modWT1, KRASG12D and KRAS G12V by HCT-116 (FIGS. 79B-C) and IFNγ responses to TBXT(FIG. 79G), WT1 (FIG. 79H), KRAS G12D (FIG. 79I) and KRAS G12D (FIG.79J) in CRC-vaccine B.

FIG. 80 shows expression of membrane bound CD40L by the CRC vaccinecomponent cell lines.

FIGS. 81 A-C show antigen specific IFNγ responses induced by the unitdose of the CRC vaccine (FIG. 81A), CRC vaccine-A (FIG. 81B) and CRCvaccine-B (FIG. 81C) compared to unmodified controls.

FIG. 82 shows antigen specific IFNγ responses induced by the unit doseof the CRC vaccine in individual donors compared to unmodified controls.

FIG. 83 shows antigen specific IFNγ responses induced by CRC vaccinecell lines alone and in cocktails of cell lines.

FIG. 84 shows endogenous expression of PCa antigens in candidate andfinal PCa vaccine cell line components.

FIGS. 85 A and B show antigens expressed by the PCa vaccine in PCapatient tumors (FIG. 85A) and the number of PCa antigens expressed bythe vaccine cell lines also expressed in PCa patient tumors (FIG. 85B).

FIGS. 86 A-D show IFNγ responses elicited by individual PCa candidatevaccine cell lines alone (FIG. 86A) and in cocktails (FIGS. 86B-C) ofcell lines and that unmodified LNCaP, NEC8, and NTERA-2c1-D1 cell linesare more immunogenic in cocktails (FIG. 86D)

FIGS. 87 A-F show the expression of and IFNγ responses to antigensintroduced in the PCa vaccine cell lines compared to unmodifiedcontrols. Expression of modTBXT (FIG. 87A) by PC3 and IFNγ responses toTBXT (FIG. 87D) in PCa-vaccine A. Expression of modMAGEC2 (FIG. 87B) byPC3 and IFNγ responses to MAGEC2 (FIG. 87E) in PCa-vaccine A. Expressionof modPSMA (FIG. 87C) by DU145 and IFNγ responses to PSMA (FIG. 87F) inPCa-vaccine B.

FIG. 88 shows expression of membrane bound CD40L by the PCa vaccinecomponent cell lines.

FIGS. 89 A-C show antigen specific IFNγ responses induced by the unitdose of the PCa vaccine (FIG. 89A), PCa vaccine-A (FIG. 89B) and PCavaccine-B (FIG. 89C) compared to unmodified controls.

FIG. 90 shows antigen specific IFNγ responses induced by the unit doseof the PCa vaccine in individual donors compared to unmodified controls.

FIGS. 91 A-E show the Pca vaccine cell lines as cocktails of cell linesare more immunogenic than single cell lines.

FIG. 91A shows IFNγ responses to individual PCA vaccine-A cell lines.Pca vaccine-A (FIG. 91B and FIG. 91D) and PCa vaccine-B (FIG. 91C andFIG. 91E) induce more robust IFNγ responses than single component celllines to parental cell lines and PCa antigens.

FIGS. 92 A and B show endogenous expression of bladder cancer antigens(FIG. 92A) and bladder cancer CSC-like markers (FIG. 92B) by candidateUBC vaccine cell lines.

FIGS. 93 A-C show IFNγ responses elicited by individual UBC candidatevaccine cell lines alone (FIG. 93A) and in cocktails (FIG. 93B and FIG.93C).

FIGS. 94 A-C show endogenous expression of bladder cancer antigens byUBC vaccine cell lines (94A), expression of these antigens patienttumors (FIG. 94B) and the number of bladder cancer antigens expressed bythe UBC vaccine cell lines also expressed in bladder cancer patienttumors (FIG. 94C).

FIGS. 95 A-H show the expression of and IFNγ responses to antigensintroduced in the UBC vaccine cell lines compared to unmodifiedcontrols. Expression of modPSMA (FIG. 95A) and modCriptol (FIG. 95B) byJ82 and IFNγ responses to PSMA (FIG. 95E) and Criptol (FIG. 95F) inducedby UBC-vaccine A. Expression of modWT1 (FIG. 95C) and modFOLR1 (FIG.95D) by SCaBER and IFNγ responses to WT1 (FIG. 95G) and FOLR1 (FIG. 95H)in UBC-vaccine B.

FIG. 96 shows expression of membrane bound CD40L by the UBC vaccinecomponent cell lines.

FIGS. 97 A-C show antigen specific IFNγ responses induced by the unitdose of the UBC vaccine (FIG. 97A), UBC vaccine-A (FIG. 97B), and UBCvaccine-B (FIG. 97C) compared to unmodified controls.

FIG. 98 shows antigen specific IFNγ responses induced by the unit doseof the UBC vaccine in individual donors compared to unmodified controls.

FIGS. 99 A and B show endogenous expression of ovarian cancer antigens(FIG. 99A) and ovarian cancer CSC-like markers (FIG. 99B) by candidateovarian cancer vaccine component cell lines.

FIGS. 100 A-C show IFNγ responses elicited by individual OC candidatevaccine cell lines alone (FIG. 100A) and in cocktails (FIG. 100B andFIG. 100C).

FIGS. 101 A-C show endogenous antigen expression by selected OC vaccinecomponent cell lines (FIG. 101A) expression of these antigens patienttumors (FIG. 101B) and the number of ovarian cancer antigens expressedby the OC vaccine cell lines also expressed in ovarian cancer patienttumors (FIG. 101C).

FIGS. 102 A-L show the expression of and IFNγ responses to antigensintroduced in the OC vaccine cell lines compared to unmodified controls.Expression of modTERT (FIG. 102A) by MCAS and IFNγ responses to TERT byOC-vaccine A (FIG. 102G), expression of modFSHR (FIG. 102B) andmodMAGEA10 (FIG. 102D) by TOV-112D and IFNγ responses to FSHR (FIG.102H) and MAGEA10 (FIG. 102I) by OC-vaccine A. Expression of modWT1(FIG. 102C) and modFOLR1 (FIG. 102E) by TOV-21G and IFNγ responses toWT1 (FIG. 102K) and FOLR1 (FIG. 102J) by OC vaccine-B. Expression ofmodBORIS by ES-2 (FIG. 102F) and IFNγ responses to BORIS by OC vaccine-B(FIG. 102L).

FIGS. 103 A and B show IFNγ responses to the unmodified and vaccinecomponent cell lines TOV-21G (FIG. 103A) and ES-2 (FIG. 103B) celllines.

FIG. 104 shows expression of membrane bound CD40L by the OC vaccinecomponent cell lines.

FIGS. 105 A-C show antigen specific IFNγ responses induced by the unitdose of the OC vaccine (FIG. 105A), OC vaccine-A (FIG. 105B), and OCvaccine-B (FIG. 105C) compared to unmodified controls.

FIG. 106 shows antigen specific IFNγ responses induced by the unit doseof the OC vaccine in individual donors compared to unmodified controls.

FIGS. 107 A and B show endogenous expression of head and neck cancerantigens (FIG. 107A) and of head and neck cancer CSC-like markers (FIG.107B) by candidate and selected head and neck cancer vaccine componentcell lines.

FIGS. 108 A and B show expression of antigens in patient tumors alsoexpressed by selected HN vaccine component cell lines (FIG. 108A) andthe number of head and neck cancer antigens expressed by the HN vaccinecell lines also expressed in head and neck cancer patient tumors (FIG.108B).

FIGS. 109 A-E show IFNγ responses elicited by individual HN candidatevaccine cell lines alone (FIG. 109A), and in cocktails of cell lines(FIG. 109B and FIG. 109C), most HN cell lines are more immunogenic incocktails (FIG. 109D), and the modified HN vaccine component cell linesare more immunogenic than the parental cell lines (FIG. 109E).

FIGS. 110 A-K show expression of modPSMA by HSC-4 (FIG. 110A) and IFNγresponses to PSMA (FIG. 110E), expression of modPRAME (FIG. 110B) andmodTBXT (FIG. 110C) by HO-1-N-1 (FIG. 110A) and IFNγ responses to PRAME(FIG. 110F) and TBXT (FIG. 110G), expression of HPV16 and HPV18 E6 andE7 by KON (FIG. 110D) and IFNγ responses to HPV16 E6 and E7 in alldonors (FIG. 110H) and individual donors (FIG. 110I), and IFNγ responsesto HPV18 E6 and E7 in all donors (FIG. 110J) and individual donors (FIG.110K).

FIG. 111 shows expression of membrane bound CD40L by the HN vaccinecomponent cell lines.

FIGS. 112 A-F show antigen specific IFNγ responses induced by the unitdose of the HN vaccine (FIG. 112A) all HN antigens and non-viral HNantigens (FIG. 112D), HN vaccine-A (FIG. 112B) to all HN antigens and tonon-viral HN antigens (FIG. 112E) and HN vaccine-B to all HN antigens(FIG. 112C) and non-viral HN antigens (FIG. 112F) compared to unmodifiedcontrols.

FIGS. 113 A and B show antigen specific responses in individual donorsto all HN antigens (top panel) and to non-viral HN antigens (bottompanel).

FIGS. 114 A and B show endogenous expression of gastric cancer antigens(FIG. 114A) and gastric cancer CSC-like markers (FIG. 114B) by candidateovarian cancer vaccine component cell lines.

FIGS. 115 A-C show IFNγ responses elicited by individual GCA candidatevaccine cell lines alone (FIG. 115A) and in cocktails (FIG. 115B andFIG. 115C).

FIGS. 116 A-C show endogenous antigen expression by selected GCA vaccinecomponent cell lines (FIG. 116A) expression of these antigens patienttumors (FIG. 116B) and the number of gastric cancer antigens expressedby the GCA vaccine cell lines also expressed in gastric cancer patienttumors (FIG. 116C).

FIGS. 117 A-H show expression of modPSMA (FIG. 117A) and modLY6K (FIG.117B) by MKN-1 and IFNγ responses to PSMA (FIG. 117E) and LY6K (FIG.117F), show expression of modWT1 (FIG. 117C) and modCLDN18 (FIG. 117D)by Fu97 and IFNγ responses to WT1 (FIG. 117G) and CLDN18 (FIG. 117H).

FIG. 118 shows expression of membrane bound CD40L by the GCA vaccinecomponent cell lines.

FIGS. 119 A-C show antigen specific IFNγ responses induced by the unitdose of the GCA vaccine (FIG. 119A), GCA vaccine-A (FIG. 119B), and GCAvaccine-B (FIG. 119C) compared to unmodified controls.

FIG. 120 shows antigen specific IFNγ responses induced by the unit doseof the GCA vaccine in individual donors compared to unmodified controls.

FIGS. 121 A and B show endogenous expression of breast cancer antigens(FIG. 121A) and breast cancer CSC-like markers (FIG. 121B) by candidatebreast cancer vaccine component cell lines.

FIGS. 122 A-D show IFNγ responses elicited by individual BRC candidatevaccine cell lines alone (FIG. 122A and FIG. 122C) and in cocktails(FIG. 122B, FIG. 122C, and FIG. 122D).

FIGS. 123 A-C show endogenous antigen expression by selected BRC vaccinecomponent cell lines (FIG. 123A) expression of these antigens in patienttumors (FIG. 123B) and breast cancer patient tumors (FIG. 123C).

FIGS. 124 A-H show expression of modPSMA by CAMA-1 (FIG. 124A) and IFNγresponses to PSMA (FIG. 124E), show expression of modTERT by AU565 (FIG.124B) and IFNγ responses to TERT (FIG. 124F), and show expression ofmodTBXT (FIG. 124C) and ModBORIS (FIG. 124D) by T47D and IFNγ responsesto TBXT (FIG. 124G) and BORIS (FIG. 124H).

FIG. 125 shows expression of membrane bound CD40L by the BRC vaccinecomponent cell lines.

FIGS. 126 A-C show antigen specific IFNγ responses induced by the unitdose of the BRC vaccine (FIG. 126A), BRC vaccine-A (FIG. 126B) and BRCvaccine-B (FIG. 126C) compared to unmodified controls.

FIG. 127 shows antigen specific IFNγ responses induced by the unit doseof the BRC vaccine in individual donors compare to unmodified controls.

FIGS. 128 A-D show BRC vaccine-A (FIG. 128A and FIG. 128C) and BRCvaccine-B (FIG. 128B and FIG. 128D) compositions induce a greaterbreadth and magnitude of antigen specific responses compared to singlecomponent cell lines.

FIG. 129 shows the sequence alignment between human native PSMA (huPSMA;SEQ ID NO: 70) and the designed PSMA with non-synonymous mutations(NSMs) (PSMAmod; SEQ ID NO: 38).

FIG. 130 A-C shows HLA supertype frequency pairs in a population.

FIG. 131 shows the number of neoepitopes existing in the cell lines of avaccine composition and designed neoepitopes in GBM recognized by donorsexpressing HLA-A and HLA-B supertype pairs within the population subsetsdescribed in FIG. 131.

FIG. 132 shows the number of neoepitopes targeted by four different mRNAimmunotherapies.

DETAILED DESCRIPTION

Embodiments of the present disclosure provide a platform approach tocancer vaccination that provides both breadth, in terms of the types ofcancer amenable to treatment by the compositions, methods, and regimensdisclosed, and magnitude, in terms of the immune responses elicited bythe compositions, methods, and regimens disclosed.

In various embodiments of the present disclosure, intradermal injectionof an allogenic whole cancer cell vaccine induces a localizedinflammatory response recruiting immune cells to the injection site.Without being bound to any theory or mechanism, following administrationof the vaccine, antigen presenting cells (APCs) that are present locallyin the skin (vaccine microenvironment, VME), such as Langerhans cells(LCs) and dermal dendritic cells (DCs), uptake vaccine cell componentsby phagocytosis and then migrate through the dermis to a draining lymphnode. At the draining lymph node, DCs or LCs that have phagocytized thevaccine cell line components can prime naïve T cells and B cells.Priming of naïve T and B cells initiates an adaptive immune response totumor associated antigens (TAAs) expressed by the vaccine cell lines. Insome embodiments of the present disclosure, the priming occurs in vivoand not in vitro or ex vivo. In embodiments of the vaccine compositionsprovided herein, the multitude of TAAs expressed by the vaccine celllines are also expressed a subject's tumor. Expansion of antigenspecific T cells at the draining lymph node and the trafficking of theseT cells to the tumor microenvironment (TME) can initiate avaccine-induced anti-tumor response.

Immunogenicity of an allogenic vaccine can be enhanced through geneticmodifications of the cell lines comprising the vaccine composition tointroduce TAAs (native/wild-type or designed/mutated as describedherein). Immunogenicity of an allogenic vaccine can be further enhancedthrough genetic modifications of the cell lines comprising the vaccinecomposition to reduce expression of immunosuppressive factors and/orincrease the expression or secretion of immunostimulatory signals.Modulation of these factors can enhance the uptake of vaccine cellcomponents by LCs and DCs in the dermis, facilitate the trafficking ofDCs and LCs to the draining lymph node, and enhance effector T cell andB cell priming in the draining lymph node, thereby providing more potentanti-tumor responses.

In various embodiments, the present disclosure provides an allogeneicwhole cell cancer vaccine platform that includes compositions andmethods for treating cancer, and/or preventing cancer, and/orstimulating an immune response. Criteria and methods according toembodiments of the present disclosure include without limitation: (i)criteria and methods for cell line selection for inclusion in a vaccinecomposition, (ii) criteria and methods for combining multiple cell linesinto a therapeutic vaccine composition, (iii) criteria and methods formaking cell line modifications, and (iv) criteria and methods foradministering therapeutic compositions with and without additionaltherapeutic agents. In some embodiments, the present disclosure providesan allogeneic whole cell cancer vaccine platform that includes, withoutlimitation, administration of multiple cocktails comprising combinationsof cell lines that together comprise one unit dose, wherein unit dosesare strategically administered over time, and additionally optionallyincludes administration of other therapeutic agents such ascyclophosphamide and additionally optionally a checkpoint inhibitor.

The present disclosure provides, in some embodiments, compositions andmethods for tailoring a treatment regimen for a subject based on thesubject's tumor type. In some embodiments, the present disclosureprovides a cancer vaccine platform whereby allogeneic cell line(s) areidentified and optionally modified and administered to a subject. Invarious embodiments, the tumor origin (primary site) of the cellline(s), the amount and number of TAAs expressed by the cell line(s),the number of cell line modifications, and the number of cell linesincluded in a unit dose are each customized based on the subject's tumortype, stage of cancer, and other considerations As described herein, thetumor origin of the cell lines may be the same or different than thetumor intended to be treated. In some embodiments, the cancer cell linesmay be cancer stem cell lines.

Definitions

In this disclosure, “comprises”, “comprising”, “containing”, “having”,and the like have the meaning ascribed to them in U.S. patent law andmean “includes”, “including”, and the like; the terms “consistingessentially of” or “consists essentially” likewise have the meaningascribed in U.S. patent law and these terms are open-ended, allowing forthe presence of more than that which is recited so long as basic ornovel characteristics of that which is recited are not changed by thepresence of more than that which is recited, but excluding prior artembodiments.

Unless specifically otherwise stated or obvious from context, as usedherein, the terms “a”, “an”, and “the” are understood to be singular orplural.

The terms “cell”, “cell line”, “cancer cell line”, “tumor cell line”,and the like as used interchangeably herein refers to a cell line thatoriginated from a cancerous tumor as described herein, and/or originatesfrom a parental cell line of a tumor originating from a specificsource/organ/tissue. In some embodiments the cancer cell line is acancer stem cell line as described herein. In certain embodiments, thecancer cell line is known to express or does express multipletumor-associated antigens (TAAs) and/or tumor specific antigens (TSAs).In some embodiments of the disclosure, a cancer cell line is modified toexpress, or increase expression of, one or more TAAs. In certainembodiments, the cancer cell line includes a cell line following anynumber of cell passages, any variation in growth media or conditions,introduction of a modification that can change the characteristics ofthe cell line such as, for example, human telomerase reversetranscriptase (hTERT) immortalization, use of xenografting techniquesincluding serial passage through xenogenic models such as, for example,patient-derived xenograft (PDX) or next generation sequencing (NGS)mice, and/or co-culture with one or more other cell lines to provide amixed population of cell lines. As used herein, the term “cell line”includes all cell lines identified as having any overlap in profile orsegment, as determined, in some embodiments, by Short Tandem Repeat(STR) sequencing, or as otherwise determined by one of skill in the art.As used herein, the term “cell line” also encompasses any geneticallyhomogeneous cell lines, in that the cells that make up the cell line(s)are clonally derived from a single cell such that they are geneticallyidentical. This can be accomplished, for example, by limiting dilutionsubcloning of a heterogeneous cell line. The term “cell line” alsoencompasses any genetically heterogeneous cell line, in that the cellsthat make up the cell line(s) are not expected to be geneticallyidentical and contain multiple subpopulations of cancer cells. Variousexamples of cell lines are described herein. Unless otherwisespecifically stated, the term “cell line” or “cancer cell line”encompasses the plural “cell lines.”

As used herein, the term “tumor” refers to an accumulation or mass ofabnormal cells. Tumors may be benign (non-cancerous), premalignant(pre-cancerous, including hyperplasia, atypia, metaplasia, dysplasia andcarcinoma in situ), or malignant (cancerous). It is well known thattumors may be “hot” or “cold”. By way of example, melanoma and lungcancer, among others, demonstrate relatively high response rates tocheckpoint inhibitors and are commonly referred to as “hot” tumors.These are in sharp contrast to tumors with low immune infiltrates called“cold” tumors or non-T-cell-inflamed cancers, such as those from theprostate, pancreas, glioblastoma, and bladder, among others. In someembodiments, the compositions and methods provided herein are useful totreat or prevent cancers with associated hot tumors. In someembodiments, the compositions and methods provided herein are useful totreat or prevent cancers with cold tumors. Embodiments of the vaccinecompositions of the present disclosure can be used to convert cold(i.e., treatment-resistant or refractory) cancers or tumors to hot(i.e., amenable to treatment, including a checkpoint inhibition-basedtreatment) cancers or tumors. Immune responses against cold tumors aredampened because of the lack of neoepitopes associated with lowmutational burden. In various embodiments, the compositions describedherein comprise a multitude of potential neoepitopes arising frompoint-mutations that can generate a multitude of exogenous antigenicepitopes. In this way, the patients' immune system can recognize theseepitopes as non-self, subsequently break self-tolerance, and mount ananti-tumor response to a cold tumor, including induction of an adaptiveimmune response to wide breadth of antigens (See Leko, V. et al. JImmunol (2019)).

Cancer stem cells are responsible for initiating tumor development, cellproliferation, and metastasis and are key components of relapsefollowing chemotherapy and radiation therapy. In certain embodiments, acancer stem cell line or a cell line that displays cancer stem cellcharacteristics is included in one or more of the vaccine compositions.As used herein, the phrase “cancer stem cell” (CSC) or “cancer stem cellline” refers to a cell or cell line within a tumor that possesses thecapacity to self-renew and to cause the heterogeneous lineages of cancercells that comprise the tumor. CSCs are highly resistant to traditionalcancer therapies and are hypothesized to be the leading driver ofmetastasis and tumor recurrence. To clarify, a cell line that displayscancer stem cell characteristics is included within the definition of a“cancer stem cell”. Exemplary cancer stem cell markers identified byprimary tumor site are provided in Table 2 and described herein. Celllines expressing one or more of these markers are encompassed by thedefinition of “cancer stem cell line”. Exemplary cancer stem cell linesare described herein, each of which are encompassed by the definition of“cancer stem cell line”.

As used herein, the phrase “each cell line or a combination of celllines” refers to, where multiple cell lines are provided in acombination, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more or the combination ofthe cell lines. As used herein, the phrase “each cell line or acombination of cell lines have been modified” refers to, where multiplecell lines are provided in combination, modification of one, some, orall cell lines, and also refers to the possibility that not all of thecell lines included in the combination have been modified. By way ofexample, the phrase “a composition comprising a therapeuticallyeffective amount of at least 2 cancer cell lines, wherein each cell lineor a combination of the cell lines comprises cells that have beenmodified . . . ” means that each of the two cell lines has been modifiedor one of the two cell lines has been modified. By way of anotherexample, the phrase “a composition comprising a therapeuticallyeffective amount of at least 3 cancer cell lines, wherein each cell lineor a combination of the cell lines comprises cells that have beenmodified . . . ” means that each (i.e., all three) of the cell lineshave been modified or that one or two of the three cell lines have beenmodified.

The term “oncogene” as used herein refers to a gene involved intumorigenesis. An oncogene is a mutated gene that contributes to thedevelopment of a cancer. In their normal, unmutated state, onocgenes arecalled proto-oncogenes, and they play roles in the regulation of celldivision.

As used herein, the phrase “identifying one or more . . . mutations,”for example in the process for preparing compositions useful forstimulating an immune response or treating cancer as described herein,refers to newly identifying, identifying within a database or dataset orotherwise using a series of criteria or one or more components thereofas described herein and, optionally, selecting the oncogene or mutationfor use or inclusion in a vaccine composition as described herein.

The phrase “ . . . cells that express at least [ ] tumor associatedantigens (TAAs) associated with a cancer of a subject intended toreceive said composition . . . ” as used herein refers to cells thatexpress, either natively or by way of genetic modification, thedesignated number of TAAs and wherein said same TAAs are expressed orknown to be expressed by cells of a patient's tumor. The expression ofspecific TAAs by cells of a patient's tumor may be determined by assay,surgical procedures (e.g., biopsy), or other methods known in the art.In other embodiments, a clinician may consult the Cancer Cell LineEncyclopedia (CCLE) and other known resources to identify a list of TAAsknown to be expressed by cells of a particular tumor type.

As used herein, the phrase “ . . . that is either not expressed orminimally expressed . . . ” means that the referenced gene or protein(e.g., a TAA or an immunosuppressive protein or an immunostimulatoryprotein) is not expressed by a cell line or is expressed at a low level,where such level is inconsequential to or has a limited impact onimmunogenicity. For example, it is readily appreciated in the art that aTAA may be present or expressed in a cell line in an amount insufficientto have a desired impact on the therapeutic effect of a vaccinecomposition including said cell line. In such a scenario, the presentdisclosure provides compositions and methods to increase expression ofsuch a TAA.

As used herein, the term “equal” generally means the same value+/−10%.In some embodiments, a measurement, such as number of cells, etc., canbe +/−1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. Similarly, as used herein andas related to amino acid position or nucleotide position, the term“approximately” refers to within 1, 2, 3, 4, or 5 such residues. Withrespect to the number of cells, the term “approximately” refers to +/−1,2, 3, 4, 5, 6, 7, 8, 9, or 10%.

As used herein, the phrase “ . . . wherein said composition is capableof stimulating a 1.3-fold increase in IFNγ production compared tounmodified cancer cell lines . . . ” means, when compared to acomposition of the same cell line or cell lines that has/have not beenmodified, the composition comprising a modified cell line or modifiedcell lines is capable of stimulating at least 1.3-fold more IFNγproduction. In this example, “at least 1.3” means 1.3, 1.4, 1.5, etc.,or higher. This definition is used herein with respect to other valuesof IFNγ production, including, but not limited to, 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 4.0, or 5.0-fold or higher increase in IFNγ productioncompared to unmodified cancer cell lines (e.g., a modified cell linecompared to an modified cell line, a composition of 2 or 3 modified celllines (e.g., a vaccine composition) compared cell lines to the samecomposition comprising unmodified cell lines, or a unit dose comprising6 modified cell lines compared to the same unit dose comprisingunmodified cell lines). In other embodiments, the IFNγ production isincreased by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25-fold or highercompared to unmodified cancer cell lines. Similarly, in variousembodiments, the present disclosure provides compositions of modifiedcells or cell lines that are compared to unmodified cells or cell lineson the basis of TAA expression, immunostimulatory factor expression,immunosuppressive factor expression, and/or immune response stimulationusing the methods provided herein and the methods known in the artincluding, but not limited to, ELISA, IFNγ ELISpot, and flow cytometry.

As used herein, the phrase “fold increase” refers to the change in unitsof expression or units of response relative to a control. By way ofexample, ELISA fold change refers to the level of secreted proteindetected for the modified cell line divided by the level of secretedprotein detected, or the lower limit of detection, by the unmodifiedcell line. In another example, fold change in expression of an antigenby flow cytometry refers to the mean fluorescence intensity (MFI) ofexpression of the protein by a modified cell line divided by the MFI ofthe protein expression by the unmodified cell line. IFNγ ELISpot foldchange refers to the average IFNγ spot-forming units (SFU) inducedacross HLA diverse donors by the test variable divided by the averageIFNγ SFU induced by the control variable. For example, the average totalantigen specific IFNγ SFU across donors by a composition of threemodified cell lines divided by the IFNγ SFU across the same donors by acomposition of the same three unmodified cell lines.

In some embodiments, the fold increase in IFNγ production will increaseas the number of modifications (e.g., the number of immunostimulatoryfactors and the number of immunosuppressive factors) is increased ineach cell line. In some embodiments, the fold increase in IFNγproduction will increase as the number of cell lines (and thus, thenumber of TAAs), whether modified or unmodified, is increased. The foldincrease in IFNγ production, in some embodiments, is thereforeattributed to the number of TAAs and the number of modifications.

As used herein, the term “modified” means genetically modified toexpress, overexpress, increase, decrease, or inhibit the expression ofone or more protein or nucleic acid. As described herein, exemplaryproteins include, but are not limited to immunostimulatory factors.Exemplary nucleic acids include sequences that can be used to knockdown(KD) (i.e., decrease expression of) or knockout (KO) (i.e., completelyinhibit expression of) immunosuppressive factors. As used herein, theterm “decrease” is synonymous with “reduce” or “partial reduction” andmay be used in association with gene knockdown. Likewise, the term“inhibit” is synonymous with “complete reduction” and may be used in thecontext of a gene knockout to describe the complete excision of a genefrom a cell.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

As used herein, the terms “patient”, “subject”, “recipient”, and thelike are used interchangeably herein to refer to any mammal, includinghumans, non-human primates, domestic and farm animals, and otheranimals, including, but not limited to dogs, horses, cats, cattle,sheep, pigs, mice, rats, and goats. Exemplary subjects are humans,including adults, children, and the elderly. In some embodiments, thesubject can be a donor.

The terms “treat”, “treating”, “treatment”, and the like, as usedherein, unless otherwise indicated, refers to reversing, alleviating,inhibiting the process of disease, disorder or condition to which suchterm applies, or one or more symptoms of such disease, disorder orcondition and includes the administration of any of the compositions,pharmaceutical compositions, or dosage forms described herein, toprevent the onset of the symptoms or the complications, alleviate thesymptoms or the complications, or eliminate the disease, condition, ordisorder. As used herein, treatment can be curative or ameliorating.

As used herein, “preventing” means preventing in whole or in part,controlling, reducing, or halting the production or occurrence of thething or event to which such term applies, for example, a disease,disorder, or condition to be prevented.

Embodiments of the methods and compositions provided herein are usefulfor preventing a tumor or cancer, meaning the occurrence of the tumor isprevented or the onset of the tumor is significantly delayed. In someembodiments, the methods and compositions are useful for treating atumor or cancer, meaning that tumor growth is significantly inhibited asdemonstrated by various techniques well-known in the art such as, forexample, by a reduction in tumor volume. Tumor volume may be determinedby various known procedures, (e.g., obtaining two dimensionalmeasurements with a dial caliper). Preventing and/or treating a tumorcan result in the prolonged survival of the subject being treated.

As used herein, the term “stimulating”, with respect to an immuneresponse, is synonymous with “promoting”, “generating”, and “eliciting”and refers to the production of one or more indicators of an immuneresponse. Indicators of an immune response are described herein. Immuneresponses may be determined and measured according to the assaysdescribed herein and by methods well-known in the art.

The phrases “therapeutically effective amount”, “effective amount”,“immunologically effective amount”, “anti-tumor effective amount”, andthe like, as used herein, indicate an amount necessary to administer toa subject, or to a cell, tissue, or organ of a subject, to achieve atherapeutic effect, such as an ameliorating or a curative effect. Thetherapeutically effective amount is sufficient to elicit the biologicalor medical response of a cell, tissue, system, animal, or human that isbeing sought by a researcher, veterinarian, medical doctor, clinician,or healthcare provider. For example, a therapeutically effective amountof a composition is an amount of cell lines, whether modified orunmodified, sufficient to stimulate an immune response as describedherein. In certain embodiments, a therapeutically effective amount of acomposition is an amount of cell lines, whether modified or unmodified,sufficient to inhibit the growth of a tumor as described herein.Determination of the effective amount or therapeutically effectiveamount is, in certain embodiments, based on publications, data or otherinformation such as, for example, dosing regimens and/or the experienceof the clinician.

The terms “administering”, “administer”, “administration”, and the like,as used herein, refer to any mode of transferring, delivering,introducing, or transporting a therapeutic agent to a subject in need oftreatment with such an agent. Such modes include, but are not limitedto, oral, topical, intravenous, intraarterial, intraperitoneal,intramuscular, intratumoral, intradermal, intranasal, and subcutaneousadministration.

As used herein, the term “vaccine composition” refers to any of thevaccine compositions described herein containing one or more (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, or more) cell lines. As described herein, oneor more of the cell lines in the vaccine composition may be modified. Incertain embodiments, one or more of the cell lines in the vaccinecomposition may not be modified. The terms “vaccine”, “tumor cellvaccine”, “cancer vaccine”, “cancer cell vaccine”, “whole cancer cellvaccine”, “vaccine composition”, “composition”, “cocktail”, “vaccinecocktail”, and the like are used interchangeably herein. In someembodiments, the vaccine compositions described herein are useful totreat or prevent cancer. In some embodiments, the vaccine compositionsdescribed herein are useful to stimulate or elicit an immune response.In such embodiments, the term “immunogenic composition” is used. In someembodiments, the vaccine compositions described herein are useful as acomponent of a therapeutic regimen to increase immunogenicity of saidregimen.

The terms “dose” or “unit dose” as used interchangeably herein refer toone or more vaccine compositions that comprise therapeutically effectiveamounts of one more cell lines. As described herein, a “dose” or “unitdose” of a composition may refer to 1, 2, 3, 4, 5, or more distinctcompositions or cocktails. In some embodiments, a unit dose of acomposition refers to 2 distinct compositions administered substantiallyconcurrently (i.e., immediate series). In exemplary embodiments, onedose of a vaccine composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10separate vials, where each vial comprises a cell line, and where celllines, each from a separate vial, are mixed prior to administration. Insome embodiments, a dose or unit dose includes 6 vials, each comprisinga cell line, where 3 cell lines are mixed and administered at one site,and the other 3 cell lines are mixed and administered at a second site.Subsequent “doses” may be administered similarly. In still otherembodiments, administering 2 vaccine cocktails at 2 sites on the body ofa subject for a total of 4 concurrent injections is contemplated.

As used herein, the term “cancer” refers to diseases in which abnormalcells divide without control and are able to invade other tissues. Thus,as used herein, the phrase “ . . . associated with a cancer of asubject” refers to the expression of tumor associated antigens,neoantigens, or other genotypic or phenotypic properties of a subject'scancer or cancers. TAAs associated with a cancer are TAAs that expressedat detectable levels in a majority of the cells of the cancer.Expression level can be detected and determined by methods describedherein. There are more than 100 different types of cancer. Most cancersare named for the organ or type of cell in which they start; forexample, cancer that begins in the colon is called colon cancer; cancerthat begins in melanocytes of the skin is called melanoma. Cancer typescan be grouped into broader categories. In some embodiments, cancers maybe grouped as solid (i.e., tumor-forming) cancers and liquid (e.g.,cancers of the blood such as leukemia, lymphoma and myeloma) cancers.Other categories of cancer include: carcinoma (meaning a cancer thatbegins in the skin or in tissues that line or cover internal organs, andits subtypes, including adenocarcinoma, basal cell carcinoma, squamouscell carcinoma, and transitional cell carcinoma); sarcoma (meaning acancer that begins in bone, cartilage, fat, muscle, blood vessels, orother connective or supportive tissue); leukemia (meaning a cancer thatstarts in blood-forming tissue (e.g., bone marrow) and causes largenumbers of abnormal blood cells to be produced and enter the blood;lymphoma and myeloma (meaning cancers that begin in the cells of theimmune system); and central nervous system cancers (meaning cancers thatbegin in the tissues of the brain and spinal cord). The termmyelodysplastic syndrome refers to a type of cancer in which the bonemarrow does not make enough healthy blood cells (white blood cells, redblood cells, and platelets) and there are abnormal cells in the bloodand/or bone marrow. Myelodysplastic syndrome may become acute myeloidleukemia (AML). By way of non-limiting examples, the compositions andmethods described herein are used to treat and/or prevent the cancerdescribed herein, including in various embodiments, lung cancer (e.g.,non-small cell lung cancer or small cell lung cancer), prostate cancer,breast cancer, triple negative breast cancer, metastatic breast cancer,ductal carcinoma in situ, invasive breast cancer, inflammatory breastcancer, Paget disease, breast angiosarcoma, phyllodes tumor, testicularcancer, colorectal cancer, bladder cancer, gastric cancer, head and neckcancer, liver cancer, renal cancer, glioma, gliosarcoma, astrocytoma,ovarian cancer, neuroendocrine cancer, pancreatic cancer, esophagealcancer, endometrial cancer, melanoma, mesothelioma, and/orhepatocellular cancers.

Examples of carcinomas include, without limitation, giant and spindlecell carcinoma; small cell carcinoma; papillary carcinoma; squamous cellcarcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrixcarcinoma; transitional cell carcinoma; papillary transitional cellcarcinoma; adenocarcinoma; gastrinoma; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in an adenomatous polyp; adenocarcinoma, familialpolyposis coli; solid carcinoma; carcinoid tumor; branchioloalveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;non-encapsulating sclerosing carcinoma; adrenal cortical carcinoma;endometroid carcinoma; skin appendage carcinoma; apocrineadenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma;mucoepidermoid carcinoma; cystadenocarcinoma; papillarycystadenocarcinoma; papillary serous cystadenocarcinoma; mucinouscystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma;infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma;inflammatory carcinoma; Paget's disease; mammary acinar cell carcinoma;adenosquamous carcinoma; adenocarcinoma with squamous metaplasia;sertoli cell carcinoma; embryonal carcinoma; and choriocarcinoma.

Examples of sarcomas include, without limitation, glomangiosarcoma;sarcoma; fibrosarcoma; myxosarcoma; liposarcoma; leiomyosarcoma;rhabdomyosarcoma; embryonal rhabdomyo sarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; carcinosarcoma; synovial sarcoma;hemangiosarcoma; kaposi's sarcoma; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; mesenchymal chondrosarcoma;giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant;ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblasticfibrosarcoma; myeloid sarcoma; and mast cell sarcoma.

Examples of leukemias include, without limitation, leukemia; lymphoidleukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cellleukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia;monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; andhairy cell leukemia.

Examples of lymphomas and myelomas include, without limitation,malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-hodgkin's lymphomas; malignant melanoma; amelanoticmelanoma; superficial spreading melanoma; malignant melanoma in giantpigmented nevus; epithelioid cell melanoma; and multiple myeloma.

Examples of brain/spinal cord cancers include, without limitation,pinealoma, malignant; chordoma; glioma, gliosarcoma, malignant;ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillaryastrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma; andneurilemmoma, malignant.

Examples of other cancers include, without limitation, a thymoma; anovarian stromal tumor; a thecoma; a granulosa cell tumor; anandroblastoma; a leydig cell tumor; a lipid cell tumor; a paraganglioma;an extra-mammary paraganglioma; a pheochromocytoma; blue nevus,malignant; fibrous histiocytoma, malignant; mixed tumor, malignant;mullerian mixed tumor; nephroblastoma; hepatoblastoma; mesenchymoma,malignant; brenner tumor, malignant; phyllodes tumor, malignant;mesothelioma, malignant; dysgerminoma; teratoma, malignant; strumaovarii, malignant; mesonephroma, malignant; hemangioendothelioma,malignant; hemangiopericytoma, malignant; chondroblastoma, malignant;granular cell tumor, malignant; malignant histiocytosis; andimmunoproliferative small intestinal disease.

All references, patents, and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

Vaccine Compositions

The present disclosure is directed to a platform approach to cancervaccination that provides breadth, with regard to the scope of cancersand tumor types amenable to treatment with the compositions, methods,and regimens disclosed, as well as magnitude, with regard to the levelof immune responses elicited by the compositions and regimens disclosed.Embodiments of the present disclosure provide compositions comprisingcancer cell lines. In some embodiments, the cell lines have beenmodified as described herein.

The compositions of the disclosure are designed to increaseimmunogenicity and/or stimulate an immune response. For example, in someembodiments, the vaccines provided herein increase IFNγ production andthe breadth of immune responses against multiple TAAs (e.g., thevaccines are capable of targeting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more TAAs, indicating thediversity of T cell receptor (TCR) repertoire of these anti-TAA T cellprecursors. In some embodiments, the immune response produced by thevaccines provided herein is a response to more than one epitopeassociated with a given TAA (e.g., the vaccines are capable of targeting1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40 epitopes or more on a given TAA), indicating the diversity of TCRrepertoire of these anti-TAA T cell precursors.

This can be accomplished in certain embodiments by selecting cell linesthat express numerous TAAs associated with the cancer to be treated;knocking down or knocking out expression of one or moreimmunosuppressive factors that facilitates tumor cell evasion of immunesystem surveillance; expressing or increasing expression of one or moreimmunostimulatory factors to increase immune activation within thevaccine microenvironment (VME); increasing expression of one or moretumor-associated antigens (TAAs) to increase the scope of relevantantigenic targets that are presented to the host immune system,optionally where the TAA or TAAs are designed or enhanced (e.g.,modified by mutation) and comprise, for example, non-synonymousmutations (NSMs) and/or neoepitopes; administering a vaccine compositioncomprising at least 1 cancer stem cell; and/or any combination thereof.

The one or more cell lines of the vaccine composition can be modified toreduce production of more than one immunosuppressive factor (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, or more immunosuppressive factors). The one ormore cell lines of a vaccine can be modified to increase production ofmore than one immunostimulatory factor (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, or more immunostimulatory factors). The one or more cell lines ofthe vaccine composition can naturally express, or be modified to expressmore than one TAA, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40 or more TAAs.

The vaccine compositions can comprise cells from 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more cell lines. Further, as described herein, cell lines canbe combined or mixed, e.g., prior to administration. In someembodiments, production of one or more immunosuppressive factors fromone or more or the combination of the cell lines can be reduced oreliminated. In some embodiments, production of one or moreimmunostimulatory factors from one or more or the combination of thecell lines can be added or increased. In certain embodiments, the one ormore or the combination of the cell lines can be selected to express aheterogeneity of TAAs. In some embodiments, the cell lines can bemodified to increase the production of one or more immunostimulatoryfactors, TAAs, and/or neoantigens. In some embodiments, the cell lineselection provides that a heterogeneity of HLA supertypes arerepresented in the vaccine composition. In some embodiments, the cellslines are chosen for inclusion in a vaccine composition such that adesired complement of TAAs are represented.

In various embodiments, the vaccine composition comprises atherapeutically effective amount of cells from at least one cancer cellline, wherein the cell line or the combination of cell lines expressesmore than one of the TAAs of Tables 7-23. In some embodiments, a vaccinecomposition is provided comprising a therapeutically effective amount ofcells from at least two cancer cell lines, wherein each cell line or thecombination of cell lines expresses at least three, at least four, atleast five, at least six, at least seven, at least eight, at least nine,or at least ten of the TAAs of Tables 7-23. In some embodiments, avaccine composition is provided comprising a therapeutically effectiveamount of cells from at least one cancer cell line, wherein the at leastone cell line is modified to express at least one of theimmunostimulatory factors of Table 4, at least two of theimmunostimulatory factors of Table 4, or at least three of theimmunostimulatory factors of Table 4. In further embodiments, a vaccinecomposition is provided comprising a therapeutically effective amount ofcells from at least one cancer cell line, wherein each cell line orcombination of cell lines is modified to reduce at least one of theimmunosuppressive factors of Table 6, or at least two of theimmunosuppressive factors of Table 6.

In embodiments where the one or more cell lines are modified to increasethe production of one or more TAAs, the expressed TAAs may or may nothave the native coding sequence of DNA/protein. That is, expression maybe codon optimized or modified. Such optimization or modification mayenhance certain effects (e.g., may lead to reduced shedding of a TAAprotein from the vaccine cell membrane). As described herein, in someembodiments the expressed TAA protein is a designed antigen comprisingone or more nonsynonymous mutations (NSMs) identified in cancerpatients. In some embodiments, the NSMs introduces CD4, CD8, or CD4 andCD8 neoepitopes.

Any of the vaccine compositions described herein can be administered toa subject in order to treat cancer, prevent cancer, prolong survival ina subject with cancer, and/or stimulate an immune response in a subject.

Cell Lines

In various embodiments of the disclosure, the cell lines comprising thevaccine compositions and used in the methods described herein originatefrom parental cancer cell lines.

Cell lines are available from numerous sources as described herein andare readily known in the art. For example, cancer cell lines can beobtained from the American Type Culture Collection (ATCC, Manassas,Va.), Japanese Collection of Research Bioresources cell bank (JCRB,Kansas City, Mo.), Cell Line Service (CLS, Eppelheim, Germany), GermanCollection of Microorganisms and Cell Cultures (DSMZ, Braunschweig,Germany), RI KEN BioResource Research Center (RCB, Tsukuba, Japan),Korean Cell Line Bank (KCLB, Seoul, South Korea), NIH AIDS ReagentProgram (NIH-ARP/Fisher BioServices, Rockland, Md.), BioresearchCollection and Research Center (BCRC, Hsinchu, Taiwan), Interlab CellLine Collection (ICLC, Genova, Italy), European Collection ofAuthenticated Cell Cultures (ECACC, Salisbury, United Kingdom), KunmingCell Bank (KCB, Yunnan, China), National Cancer Institute DevelopmentTherapeutics Program (NCI-DTP, Bethesda, Md.), Rio de Janeiro Cell Bank(BCRJ, Rio de Janeiro, Brazil), Experimental Zooprophylactic Instituteof Lombardy and Emilia Romagna (IZSLER, Milan, Italy), Tohoku Universitycell line catalog (TKG, Miyagi, Japan), and National Cell Bank of Iran(NCBI, Tehran, Iran). In some embodiments, cell lines are identifiedthrough an examination of RNA-seq data with respect to TAAs,immunosuppressive factor expression, and/or other information readilyavailable to those skilled in the art.

In various embodiments, the cell lines in the compositions and methodsdescribed herein are from parental cell lines of solid tumorsoriginating from the lung, prostate, testis, breast, urinary tract,colon, rectum, stomach, head and neck, liver, kidney, nervous system,endocrine system, mesothelium, ovaries, pancreas, esophagus, uterus orskin. In certain embodiments, the parental cell lines comprise cells ofthe same or different histology selected from the group consisting ofsquamous cells, adenocarcinoma cells, adenosquamous cells, large cellcells, small cell cells, sarcoma cells, carcinosarcoma cells, mixedmesodermal cells, and teratocarcinoma cells. In related embodiments, thesarcoma cells comprise osteosarcoma, chondrosarcoma, leiomyosarcoma,rhabdomyosarcoma, mesothelioma, fibrosarcoma, angiosarcoma, liposarcoma,glioma, gliosarcoma, astrocytoma, myxosarcoma, mesenchymous or mixedmesodermal cells.

In certain embodiments, the cell lines comprise cancer cells originatingfrom lung cancer, non-small cell lung cancer (NSCLC), small cell lungcancer (SCLC), prostate cancer, glioblastoma, colorectal cancer, breastcancer including triple negative breast cancer (TNBC), bladder orurinary tract cancer, squamous cell head and neck cancer (SCCHN), liverhepatocellular (HCC) cancer, kidney or renal cell carcinoma (RCC)cancer, gastric or stomach cancer, ovarian cancer, esophageal cancer,testicular cancer, pancreatic cancer, central nervous system cancers,endometrial cancer, melanoma, and mesothelium cancer.

According to various embodiments, the cell lines are allogeneic celllines (i.e., cells that are genetically dissimilar and henceimmunologically incompatible, although from individuals of the samespecies.) In certain embodiments, the cell lines are geneticallyheterogeneous allogeneic. In other embodiments, the cell lines aregenetically homogeneous allogeneic.

Allogeneic cell-based vaccines differ from autologous vaccines in thatthey do not contain patient-specific tumor antigens. Embodiments of theallogeneic vaccine compositions disclosed herein compriselaboratory-grown cancer cell lines known to express TAAs of a specifictumor type. Embodiments of the allogeneic cell lines of the presentdisclosure are strategically selected, sourced, and modified prior touse in a vaccine composition. Vaccine compositions of embodiments of thepresent disclosure can be readily mass-produced. This efficiency indevelopment, manufacturing, storage, and other areas can result in costreductions and economic benefits relative to autologous-based therapies.

Tumors are typically made up of a highly heterogeneous population ofcancer cells that evolve and change over time. Therefore, it can behypothesized that a vaccine composition comprising only autologous celllines that do not target this cancer evolution and progression may beinsufficient in the elicitation of a broad immune response required foreffective vaccination. As described in embodiments of the vaccinecomposition disclosed herein, use of one or more strategically selectedallogeneic cell lines with certain genetic modification(s) addressesthis disparity.

In some embodiments, the allogeneic cell-based vaccines are from cancercell lines of the same type (e.g., breast, prostate, lung) of the cancersought to be treated. In other embodiments, various types of cell lines(i.e., cell lines from different primary tumor origins) are combined(e.g., stem cell, prostate, testes). In some embodiments, the cell linesin the vaccine compositions are a mixture of cell lines of the same typeof the cancer sought to be treated and cell lines from different primarytumor origins.

Exemplary cancer cell lines, including, but not limited to thoseprovided in Table 1, below, are contemplated for use in the compositionsand methods described herein. The Cell Line Sources identified hereinare for exemplary purposes only. The cell lines described in variousembodiments herein may be available from multiple sources.

TABLE 1 Exemplary vaccine composition cell lines per indicationAnatomical Site of Cell Line Cell Line Cell Line Source Primary TumorCommon Name Source Identification Lung ABC-1 JCRB JCRB0815 (Small Celland Calu-1 ATCC HTB-54 Non-Small Cell) LOU-NH91 DSMZ ACC-393 NCI-H1581ATCC CRL-5878 NCI-H1703 ATCC CRL-5889 NCI-H460 ATCC HTB-177 NCI-H520ATCC HTB-182 A549 ATCC CCL-185 LK-2 JCRB JCRB0829 NCI-H23 ATCC CRL-5800NCI-H2066 ATCC CRL-5917 NCI-H2009 ATCC CRL-5911 NCI-H2023 ATCC CRL-5912RERF-LC-Ad1 JCRB JCRB1020 SK-LU-1 ATCC HTB-57 NCI-H2172 ATCC CRL-5930NCI-H292 ATCC CRL-1848 NCI-H661 ATCC HTB-183 SQ-1 RCB RCB1905 RERF-LC-KJJCRB JCRB0137 SW900 ATCC HTB-59 NCI-H838 ATCC CRL-5844 NCI-H1693 ATCCCRL-5887 HCC2935 ATCC CRL-2869 NCI-H226 ATCC CRL-5826 HCC4006 ATCCCRL-2871 DMS 53 ATCC CRL-2062 DMS 114 ATCC CRL-2066 NCI-H196 ATCCCRL-5823 NCI-H1092 ATCC CRL-5855 SBC-5 JCRB JCRB0819 NCI-H510A ATCCHTB-184 NCI-H889 ATCC CRL-5817 NCI-H1341 ATCC CRL-5864 NCIH-1876 ATCCCRL-5902 NCI-H2029 ATCC CRL-5913 NCI-H841 ATCC CRL-5845 NCI-H1694 ATCCCRL-5888 DMS 79 ATCC CRL-20496 HCC33 DSMZ ACC-487 NCI-H1048 ATCCCRL-5853 NCI-H1105 ATCC CRL-5856 NCI-H1184 ATCC CRL-5858 NCI-H128 ATCCHTB-120 NCI-H1436 ATCC CRL-5871 DMS 153 ATCC CRL-2064 NCI-H1836 ATCCCRL-5898 NCI-H1963 ATCC CRL-5982 NCI-H2081 ATCC CRL-5920 NCI-H209 ATCCHTB-172 NCI-H211 ATCC CRL-524 NCI-H2171 ATCC CRL-5929 NCI-H2196 ATCCCRL-5932 NCI-H2227 ATCC CRL-5934 NCI-H446 ATCC HTB-171 NCI-H524 ATCCCRL-5831 NCI-H526 ATCC CRL-5811 NCI-H69 ATCC HTB-119 NCI-H82 ATCCHTB-175 SHP-77 ATCC CRL-2195 SW1271 ATCC CRL-2177 Prostate or PC3 ATCCCRL-1435 Testis DU145 ATCC HTB-81 LNCaP clone FGC ATCC CRL-2023 NCCITATCC CRL-2073 NEC-8 JCRB JCRB0250 NTERA-2cl-D1 ATCC CRL-1973 NCI-H660ATCC CRL-5813 VCaP ATCC CRL-2876 MDA-PCa-2b ATCC CRL-2422 22Rv1 ATCCCRL-2505 E006AA Millipore SCC102 NEC14 JCRB JCRB0162 SuSa DSMZ ACC-747833K-E ECACC 06072611 Colorectal LS123 ATCC CCL-255 HCT15 ATCC CCL-225SW1463 ATCC CCL-234 RKO ATCC CRL-2577 HUTU80 ATCC HTB-40 HCT116 ATCCCCL-247 LOVO ATCC CCL-229 T84 ATCC CCL-248 LS411N ATCC CRL-2159 SW48ATCC CCL-231 C2BBe1 ATCC CRL-2102 Caco-2 ATCC HTB-37 SNU-1033 KCLB 01033COLO 201 ATCC CCL-224 GP2d ECACC 95090714 CL-14 DSMZ ACC-504 SW403 ATCCCCL-230 SW1116 ATCC CCL-233 SW837 ATCC CCL-235 SK-CO-1 ATCC HTB-39 CL-34DSMZ ACC-520 NCI-H508 ATCC CCL-253 CCK-81 JCRB JCRB0208 SNU-C2A ATCCCCL-250.1 GP2d ECACC 95090714 HT-55 ECACC 85061105 MDST8 ECACC 99011801RCM-1 JCRB JCRB0256 CL-40 DSMZ ACC-535 COLO 678 DSMZ ACC-194 LS180 ATCCCL-187 Breast BT20 ATCC HTB-19 BT549 ATCC HTB-122 MDA-MB-231 ATCC HTB-26HS578T ATCC HTB-126 AU565 ATCC CRL-2351 CAMA1 ATCC HTB-21 MCF7 ATCCHTB-22 T-47D ATCC HTB-133 ZR-75-1 ATCC CRL-1500 MDA-MB-415 ATCC HTB-128CAL-51 DSMZ ACC-302 CAL-120 DSMZ ACC-459 HCC1187 ATCC CRL-2322 HCC1395ATCC CRL-2324 SK-BR-3 ATCC HTB-30 HDQ-P1 DSMZ ACC-494 HCC70 ATCCCRL-2315 HCC1937 ATCC CRL-2336 MDA-MB-436 ATCC HTB-130 MDA-MB-468 ATCCHTB-132 MDA-MB-157 ATCC HTB-24 HMC-1-8 JCRB JCRB0166 Hs 274.T ATCCCRL-7222 Hs 281.T ATCC CRL-7227 JIMT-1 ATCC ACC-589 Hs 343.T ATCCCRL-7245 Hs 606.T ATCC CRL-7368 UACC-812 ATCC CRL-1897 UACC-893 ATCCCRL-1902 Urinary Tract UM-UC-3 ATCC CRL-1749 5637 ATCC HTB-9 J82 ATCCHTB-1 T24 ATCC HTB-4 HT-1197 ATCC CRL-1473 TCCSUP ATCC HTB-5 HT-1376ATCC CRL-1472 SCaBER ATCC HTB-3 RT4 ATCC HTB-2 CAL-29 DSMZ ACC-515 AGSATCC CRL-1739 KMBC-2 JCRB JCRB1148 253J KCLB 080001 253J-BV KCLB 080002SW780 ATCC CRL-2169 SW1710 DSMZ ACC-426 VM-CUB-1 DSMZ ACC-400 BC-3C DSMZACC-450 U-BLC1 ECACC U-BLC1 KMBC-2 JCRB JCRB1148 RT112/84 ECACC 85061106UM-UC-1 ECACC 06080301 RT-112 DSMZ ACC-418 KU-19-19 DSMZ ACC-395 639VDSMZ ACC-413 647V DSMZ ACC-414 Kidney A-498 ATCC HTB-44 A-704 ATCCHTB-45 769-P ATCC CRL-1933 786-O ATCC CRL-1932 ACHN ATCC CRL-1611 KMRC-1JCRB JCRB1010 KMRC-2 JCRB JCRB1011 VMRC-RCZ JCRB JCRB0827 VMRC-RCW JCRBJCRB0813 UO-31 NCI-DTP UO-31 Caki-1 ATCC HTB-46 Caki-2 ATCC HTB-47OS-RC-2 RCB RCB0735 TUHR-4TKB RCB RCB1198 RCC-10RGB RCB RCB1151 SNU-1272KCLB 01272 SNU-349 KCLB 00349 TUHR-14TKB RCB RCB1383 TUHR-10TKB RCBRCB1275 BFTC-909 DSMZ ACC-367 CAL-54 DSMZ ACC-365 KMRC-3 JCRB JCRB1012KMRC-20 JCRB JCRB1071 Upper HSC-4 JCRB JCRB0624 Aerodigestive DETROIT562 ATCC CCL-138 Tract SCC-9 ATCC CRL-1629 (Head and Neck) SCC-4 ATCCCRL-1624 OSC-19 JCRB JCRB0198 KON JCRB JCRB0194 HO-1-N-1 JCRB JCRB0831OSC-20 JCRB JCRB0197 HSC-3 JCRB JCRB0623 SNU-1066 KCLB 01066 SNU-1041KCLB 01041 SNU-1076 KCLB 01076 BICR 18 ECACC 06051601 CAL-33 DSMZACC-447 YD-8 KCLB 60501 FaDu ATCC HTB-43 2A3 ATCC CRL-3212 CAL-27 ATCCCRL-2095 SCC-25 ATCC CRL-1628 SCC-15 ATCC CRL-1623 HO-1-u-1 JCRBJCRB0828 KOSC-2 JCRB JCRB0126.1 RPMI-2650 ATCC CCL-30 SCC-90 ATCCCRL-3239 SKN-3 JCRB JCRB1039 HSC-2 JCRB JCRB0622 Hs 840.T ATCC CRL-7573SAS JCRB JCRB0260 SAT JCRB JCRB1027 SNU-46 KCLB 00046 YD-38 KCLB 60508SNU-899 KCLB 00899 HN DSMZ ACC-417 BICR 10 ECACC 04072103 BICR 78 ECACC04072111 Ovaries OVCAR-3 ATCC HTB-161 TOV-112D ATCC CRL-11731 ES-2 ATCCCRL-1978 TOV-21G ATCC CRL-11730 OVTOKO JCRB JCRB1048 KURAMOCHI JCRBJCRB0098 MCAS JCRB JCRB0240 TYK-nu JCRB JCRB0234.0 OVSAHO JCRB JCRB1046OVMANA JCRB JCRB1045 JHOM-2B RCB RCB1682 OV56 ECACC 96020759 JHOS-4 RCBRCB1678 JHOC-5 RCB RCB1520 OVCAR-4 NCI-DTP OVCAR-4 JHOS-2 RCB RCB1521EFO-21 DSMZ ACC-235 OV-90 ATCC CRL-11732 OVKATE JCRB JCRB1044 SK-OV-3ATCC HTB-77 Caov-4 ATCC HTB-76 Coav-3 ATCC HTB-75 JHOM-1 RCB RCB1676COV318 ECACC 07071903 OVK-18 RCB RCB1903 SNU-119 KCLB 00119 SNU-840 KCLB00840 SNU-8 KCLB 0008 COV362 ECACC 07071910 COV434 ECACC 07071909 COV644ECACC 07071908 OV7 ECACC 96020764 OAW-28 ECACC 85101601 OVCAR-8 NCI-DTPOVCAR-8 59M ECACC 89081802 EFO-27 DSMZ ACC-191 Pancreas PANC-1 ATCCCRL-1469 HPAC ATCC CRL-2119 KP-2 JCRB JCRB0181 KP-3 JCRB JCRB0178.0 KP-4JCRB JCRB0182 HPAF-II ATCC CRL-1997 SUIT-2 JCRB JCRB1094 AsPC-1 ATCCCRL-1682 PSN1 ATCC CRL-3211 CFPAC-1 ATCC CRL-1918 Capan-1 ATCC HTB-79Panc 02.13 ATCC CRL-2554 Panc 03.27 ATCC CRL-2549 BxPC-3 ATCC CRL-1687SU.86.86 ATCC CRL-1837 Hs 766T ATCC HTB-134 Panc 10.05 ATCC CRL-2547Panc 04.03 ATCC CRL-2555 PaTu 8988s DSMZ ACC-204 PaTu 8988t DSMZ ACC-162SW1990 ATCC CRL-2172 SNU-324 KCLB 00324 SNU-213 KCLB 00213 DAN-G DSMZACC-249 Panc 02.03 ATCC CRL-2553 PaTu 8902 DSMZ ACC-179 Capan-2 ATCCHTB-80 MIA PaCa-2 ATCC CRL-1420 YAPC DSMZ ACC-382 HuP-T3 DSMZ ACC-259T3M-4 RCB RCB1021 PK-45H RCB RCB1973 Panc 08.13 ATCC CRL-2551 PK-1 RCBRCB1972 PK-59 RCB RCB1901 HuP-T4 DSMZ ACC-223 Panc 05.04 ATCC CRL-2557Stomach RERF-GC-1B JCRB JCRB1009 Fu97 JCRB JCRB1074 MKN74 JCRB JCRB0255NCI-N87 ATCC CRL-5822 NUGC-2 JCRB JCRB0821 MKN45 JCRB JCRB0254 OCUM-1JCRB JCRB0192 MKN7 JCRB JCRB1025 MKN1 JCRB JCRB0252 ECC10 RCB RCB0983TGBC-11-TKB RCB RCB1148 SNU-620 KCLB 00620 GSU RCB RCB2278 KE-39 RCBRCB1434 HuG1-N RCB RCB1179 NUGC-4 JCRB JCRB0834 SNU-16 ATCC CRL-5974SJSA-1 ATCC CRL-2098 RD-ES ATCC HTB-166 U2OS ATCC HTB-96 SaOS-2 ATCCHTB-85 Hs 746.T ATCC HTP-135 LMSU RCB RCB1062 SNU-520 KCLB 00520 GSS RCBRCB2277 ECC12 RCB RCB1009 GCIY RCB RCB0555 SH-10-TC RCB RCB1940 HGC-27BCRJ 0310 HuG1-N RCB RCB1179 SNU-601 KCLB KCLB00601 SNU-668 KCLB 00668NCC-StC-K140 JCRB JCRB1228 SNU-719 KCLB 00719 SNU-216 KCLB 00216 NUGC-3JCRB JCRB0822 Liver Hep-G2 ATCC HB-8065 JHH-2 JCRB JCRB1028 JHH-4 JCRBJCRB0435 JHH-6 JCRB JCRB1030 Li7 RCB RCB1941 HLF JCRB JCRB0405 HuH-6 RCBBRC1367 JHH-5 JCRB JCRB1029 HuH-7 JCRB JCRB0403 SNU-182 ATCC CRL-2235JHH-7 JCRB JCRB1031 SK-HEP-1 ATCC HTB-52 Hep 3B2.1-7 ATCC HB-8064SNU-449 ATCC CRL-2234 SNU-761 KCLB KCLB JHH-1 JCRB JCRB1062 SNU-398 ATCCCRL-2233 SNU-423 ATCC CRL-2238 SNU-387 ATCC CRL-2237 SNU-475 ATCCCRL-2236 SNU-886 KCLB KCLB 00886 SNU-878 KCLB KCLB 00878 NCI-H684 KCLBKCLB 90684 PLC/PRF/5 ATCC CRL-8024 HuH-1 JCRB JCRB0199 HLE JCRB JCRB0404C3A ATCC HB-8065 Central Nervous DBTRG-05MG ATCC CRL-2020 System LN-229ATCC CRL-2611 SF-126 JCRB IFO50286 M059K ATCC CRL-2365 M059KJ ATCCCRL-2366 U-251 MG JCRB IFO50288 A-172 ATCC CRL-1620 YKG-1 ATCC JCRB0746GB-1 ATCC IFO50489 KNS-60 ATCC IFO50357 KNS-81 JCRB IFO50359 TM-31 RCBRCB1731 NMC-G1 JCRB IFO50467 SNU-201 KCLB 00201 SW1783 ATCC HTB-13 GOS-3DSMZ ACC-408 KNS-81 JCRB IFO50359 KG-1-C JCRB JCRB0236 AM-38 JCRBIFO50492 CAS-1 ILCL HTL97009 H4 ATCC HTB-148 D283 Med ATCC HTB-185 DK-MGDSMZ ACC-277 U-118MG ATCC HTB-15 SNU-489 KCLB 00489 SNU-466 KCLB 00426SNU-1105 KCLB 01105 SNU-738 KCLB 00738 SNU-626 KCLB 00626 Daoy ATCCHTB-186 D341 Med ATCC HTB-187 SW1088 ATCC HTB-12 Hs 683 ATCC HTB-138ONS-76 JCRB IFO50355 LN-18 ATCC CRL-2610 T98G ATCC CRL-1690 GMS-10 DSMZACC-405 42-MG-BA DSMZ ACC-431 GaMG DSMZ ACC-242 8-MG-BA DSMZ ACC-432IOMM-Lee ATCC CRL-3370 SF268 NCI-DTP SF-268 SF539 NCI-DTP SF-539 SNB75NCI-DTP SNB-75 Esophagus TE-10 RCB RCB2099 TE-6 RCB RCB1950 TE-4 RCBRCB2097 EC-GI-10 RCB RCB0774 OE33 ECACC 96070808 TE-9 RCB RCB1988 TTJCRB JCRB0262 TE-11 RCB RCB2100 OE19 ECACC 96071721 OE21 ECACC 96062201KYSE-450 JCRB JCRB1430 TE-14 RCB RCB2101 TE-8 RCB RCB2098 KYSE-410 JCRBJCRB1419 KYSE-140 DSMZ ACC-348 KYSE-180 JCRB JCRB1083 KYSE-520 JCRBJCRB1439 KYSE-270 JCRB JCRB1087 KYSE-70 JCRB JCRB0190 TE-1 RCB RCB1894TE-5 RCB RCB1949 TE-15 RCB RCB1951 KYSE-510 JCRB JCRB1436 KYSE-30 ECACC94072011 KYSE-150 DSMZ ACC-375 COLO 680N DSMZ ACC-182 KYSE-450 JCRBJCRB1430 TE-10 RCB RCB2099 ESO-26 ECACC 11012009 ESO-51 ECACC 11012010FLO-1 ECACC 11012001 KYAE-1 ECACC 11012002 KYSE-220 JCRB JCRB1086KYSE-50 JCRB JCRB0189 OACM5.1 C ECACC 11012006 OACP4 C ECACC 11012005Endometrium SNG-M JCRB IFO50313 HEC-1-B ATCC HTB-113 JHUEM-3 Riken RCBRCB1552 RL95-2 ATCC CRL-1671 MFE-280 ECACC 98050131 MFE-296 ECACC98031101 TEN Riken RCB RCB1433 JHUEM-2 Riken RCB RCB1551 AN3-CA ATCCHTB-111 KLE ATCC CRL-1622 Ishikawa ECACC 99040201 HEC-151 JCRB JCRB1122SNU-1077 KCLB 01077 MFE-319 DSMZ ACC-423 EFE-184 DSMZ ACC-230 HEC-108JCRB JCRB1123 HEC-265 JCRB JCRB1142 HEC-6 JCRB JCRB1118 HEC-50B JCRBJCRB1145 JHUEM-1 RCB RCB1548 HEC-251 JCRB JCRB1141 COLO 684 ECACC87061203 SNU-685 KCLB 00685 HEC-59 JCRB JCRB1120 EN DSMZ ACC-564 ESS-1DSMZ ACC-461 HEC-1A ATCC HTB-112 JHUEM-7 RCB RCB1677 HEC-1 JCRB JCRB0042Skin RPMI-7951 ATCC HTB-66 MeWo ATCC HTB-65 Hs 688(A).T ATCC CRL-7425COLO 829 ATCC CRL-1974 C32 ATCC CRL-1585 A-375 ATCC CRL-1619 Hs 294TATCC HTB-140 Hs 695T ATCC HTB-137 Hs 852T ATCC CRL-7585 A2058 ATCCCRL-11147 RVH-421 DSMZ ACC-127 Hs 895.T ATCC CRL-7637 Hs 940.T ATCCCRL-7691 SK-MEL-1 ATCC HTB-67 SK-MEL-28 ATCC HTB-72 SH-4 ATCC CRL-7724COLO 800 ECACC 93051123 COLO 783 DSMZ ACC-257 MDA-MB-435S ATCC HTB-129IGR-1 CLS 300219/p483_IGR-1 IGR-39 DSMZ ACC-239 HT-144 ATCC HTB-63SK-MEL-31 ATCC HTB-73 Hs 839.T ATCC CRL-7572 Hs 600.T ATCC CRL-7360A101D ATCC CRL-7898 IPC-298 DSMZ ACC-251 SK-MEL-24 ATCC HTB-71 SK-MEL-3ATCC HTB-69 HMCB ATCC CRL-9607 Malme-3M ATCC HTB-64 Mel JuSo DSMZ ACC-74COLO 679 RCB RCB0989 COLO 741 ECACC 93052621 SK-MEL-5 ATCC HTB-70WM266-4 ATCC CRL-1676 IGR-37 DSMZ ACC-237 Hs 934.T ATCC CRL-7684UACC-257 NCI-DTP UACC-257 Mesothelium NCI-H28 ATCC CRL-5820 MSTO-211HATCC CRL-2081 IST-Mes1 ICLC HTL01005 ACC-MESO-1 RCB RCB2292 NCI-H2052ATCC CRL-5951 NCI-H2452 ATCC CRL-2081 MPP 89 ICLC HTL00012 IST-Mes2 ICLCHTL01007 RS-5 DSMZ ACC-604 DM-3 DSMZ ACC-595 JL-1 DSMZ ACC-596 COR-L321ECACC 96020756

In addition to the cell lines identified in Table 1, the following celllines are also contemplated in various embodiments.

In various embodiments, one or more non-small cell lung (NSCLC) celllines are prepared and used according to the disclosure. By way ofexample, the following NSCLC cell lines are contemplated: NCI-H460,NCIH520, A549, DMS 53, LK-2, and NCI-H23. Additional NSCLC cell linesare also contemplated by the present disclosure. As described herein,inclusion of a cancer stem cell line such as DMS 53 in a vaccinecomprising NSCLC cell lines is also contemplated.

In some embodiments, one or more prostate cancer cell lines are preparedand used according to the disclosure. By way of example, the followingprostate cancer cell lines are contemplated: PC3, DU-145, LNCAP, NEC8,and NTERA-2c1-D1. Additional prostate cancer cell lines are alsocontemplated by the present disclosure. As described herein, inclusionof a cancer stem cell line such as DMS 53 in a vaccine comprisingprostate cancer cell lines is also contemplated.

In some embodiments, one or more colorectal cancer (CRC) cell lines areprepared and used according to the disclosure. By way of example, thefollowing colorectal cancer cell lines are contemplated: HCT-15, RKO,HuTu-80, HCT-116, and LS411N. Additional colorectal cancer cell linesare also contemplated by the present disclosure. As described herein,inclusion of a cancer stem cell line such as DMS 53 in a vaccinecomprising CRC cell lines is also contemplated.

In some embodiments, one or more breast cancer or triple negative breastcancer (TNBC) cell lines are prepared and used according to thedisclosure. By way of example, the following TNBC cell lines arecontemplated: Hs 578T, AU565, CAMA-1, MCF-7, and T-47D. Additionalbreast cancer cell lines are also contemplated by the presentdisclosure. As described herein, inclusion of a cancer stem cell linesuch as DMS 53 in a vaccine comprising breast and/or TNBC cancer celllines is also contemplated.

In some embodiments, one or more bladder or urinary tract cancer celllines are prepared and used according to the disclosure. By way ofexample, the following urinary tract or bladder cancer cell lines arecontemplated: UM-UC-3, J82, TCCSUP, HT-1376, and SCaBER. Additionalbladder cancer cell lines are also contemplated by the presentdisclosure. As described herein, inclusion of a cancer stem cell linesuch as DMS 53 in a vaccine comprising bladder or urinary tract cancercell lines is also contemplated.

In some embodiments, one or more stomach or gastric cancer cell linesare prepared and used according to the disclosure. By way of example,the following stomach or gastric cancer cell lines are contemplated:Fu97, MKN74, MKN45, OCUM-1, and MKN1. Additional stomach cancer celllines are also contemplated by the present disclosure. As describedherein, inclusion of a cancer stem cell line such as DMS 53 in a vaccinecomprising stomach or gastric cancer cell lines is also contemplated.

In some embodiments, one or more squamous cell head and neck cancer(SCCHN) cell lines are prepared and used according to the disclosure. Byway of example, the following SCCHN cell lines are contemplated: HSC-4,Detroit 562, KON, HO-1-N-1, and OSC-20. Additional SCCHN cell lines arealso contemplated by the present disclosure. As described herein,inclusion of a cancer stem cell line such as DMS 53 in a vaccinecomprising SCCHN cancer cell lines is also contemplated.

In some embodiments, one or more small cell lung cancer (SCLC) celllines are prepared and used according to the disclosure. By way ofexample, the following SCLC cell lines are contemplated: DMS 114,NCI-H196, NCI-H1092, SBC-5, NCI-H510A, NCI-H889, NCI-H1341, NCIH-1876,NCI-H2029, NCI-H841, and NCI-H1694. Additional SCLC cell lines are alsocontemplated by the present disclosure. As described herein, inclusionof a cancer stem cell line such as DMS 53 in a vaccine comprising SCLCcell lines is also contemplated.

In some embodiments, one or more liver or hepatocellular cancer (HCC)cell lines are prepared and used according to the disclosure. By way ofexample, the following HCC cell lines are contemplated: Hep-G2, JHH-2,JHH-4, JHH-6, Li7, HLF, HuH-6, JHH-5, and HuH-7. Additional HCC celllines are also contemplated by the present disclosure. As describedherein, inclusion of a cancer stem cell line such as DMS 53 in a vaccinecomprising liver or HCC cancer cell lines is also contemplated.

In some embodiments, one or more kidney cancer such as renal cellcarcinoma (RCC) cell lines are prepared and used according to thedisclosure. By way of example, the following RCC cell lines arecontemplated: A-498, A-704, 769-P, 786-O, ACHN, KMRC-1, KMRC-2,VMRC-RCZ, and VMRC-RCW. Additional RCC cell lines are also contemplatedby the present disclosure. As described herein, inclusion of a cancerstem cell line such as DMS 53 in a vaccine comprising kidney or RCCcancer cell lines is also contemplated.

In some embodiments, one or more glioblastoma (GBM) cancer cell linesare prepared and used according to the disclosure. By way of example,the following GBM cell lines are contemplated: DBTRG-05MG, LN-229,SF-126, GB-1, and KNS-60. Additional GBM cell lines are alsocontemplated by the present disclosure. As described herein, inclusionof a cancer stem cell line such as DMS 53 in a vaccine comprising GBMcancer cell lines is also contemplated.

In some embodiments, one or more ovarian cancer cell lines are preparedand used according to the disclosure. By way of example, the followingovarian cell lines are contemplated: TOV-112D, ES-2, TOV-21G, OVTOKO,and MCAS. Additional ovarian cell lines are also contemplated by thepresent disclosure. As described herein, inclusion of a cancer stem cellline such as DMS 53 in a vaccine comprising ovarian cancer cell lines isalso contemplated.

In some embodiments, one or more esophageal cancer cell lines areprepared and used according to the disclosure. By way of example, thefollowing esophageal cell lines are contemplated: TE-10, TE-6, TE-4,EC-GI-10, OE33, TE-9, TT, TE-11, OE19, OE21. Additional esophageal celllines are also contemplated by the present disclosure. As describedherein, inclusion of a cancer stem cell line such as DMS 53 in a vaccinecomprising esophageal cancer cell lines is also contemplated.

In some embodiments, one or more pancreatic cancer cell lines areprepared and used according to the disclosure. By way of example, thefollowing pancreatic cell lines are contemplated: PANC-1, KP-3, KP-4,SUIT-2, and PSN1. Additional pancreatic cell lines are also contemplatedby the present disclosure. As described herein, inclusion of a cancerstem cell line such as DMS 53 in a vaccine comprising pancreatic cancercell lines is also contemplated.

In some embodiments, one or more endometrial cancer cell lines areprepared and used according to the disclosure. By way of example, thefollowing endometrial cell lines are contemplated: SNG-M, HEC-1-B,JHUEM-3, RL95-2, MFE-280, MFE-296, TEN, JHUEM-2, AN3-CA, and Ishikawa.Additional endometrial cell lines are also contemplated by the presentdisclosure. As described herein, inclusion of a cancer stem cell linesuch as DMS 53 in a vaccine comprising endometrial cancer cell lines isalso contemplated.

In some embodiments, one or more melanoma cancer cell lines are preparedand used according to the disclosure. By way of example, the followingmelanoma cell lines are contemplated: RPMI-7951, MeWo, Hs 688(A).T, COLO829, C32, A-375, Hs 294T, Hs 695T, Hs 852T, and A2058. Additionalmelanoma cell lines are also contemplated by the present disclosure. Asdescribed herein, inclusion of a cancer stem cell line such as DMS 53 ina vaccine comprising melanoma cancer cell lines is also contemplated.

In some embodiments, one or more mesothelioma cancer cell lines areprepared and used according to the disclosure. By way of example, thefollowing mesothelioma cell lines are contemplated: NCI-H28, MSTO-211H,IST-Mes1, ACC-MESO-1, NCI-H2052, NCI-H2452, MPP 89, and IST-Mes2.Additional mesothelioma cell lines are also contemplated by the presentdisclosure. As described herein, inclusion of a cancer stem cell linesuch as DMS 53 in a vaccine comprising mesothelioma cancer cell lines isalso contemplated.

Embodiments of vaccine compositions according to the disclosure are usedto treat and/or prevent various types of cancer. In some embodiments, avaccine composition may comprise cancer cell lines that originated fromthe same type of cancer. For example, a vaccine composition may comprise1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NSCLC cell lines, and such acomposition may be useful to treat or prevent NSCLC. According tocertain embodiments, the vaccine composition comprising NCSLC cell linesmay be used to treat or prevent cancers other than NSCLC, examples ofwhich are described herein.

In some embodiments, a vaccine composition may comprise cancer celllines that originated from different types of cancer. For example, avaccine composition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreNSCLC cell lines, plus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more SCLC cancercell lines, optionally plus one or other cancer cell lines, such ascancer stem cell lines, and so on, and such a composition may be usefulto treat or prevent NSCLC, and/or prostate cancer, and/or breast cancer,and so on. According to some embodiments, the vaccine compositioncomprising different cancer cell lines as described herein may be usedto treat or prevent various cancers. In some embodiments, the targetingof a TAA or multiple TAAs in a particular tumor is optimized by usingcell lines derived from different tissues or organs within a biologicalsystem to target a cancer of primary origin within the same system. Byway of non-limiting examples, cell lines derived from tumors of thereproductive system (e.g., ovaries, fallopian tubes, uterus, vagina,mammary glands, testes, vas deferens, seminal vesicles, and prostate)may be combined; cell lines derived from tumors of the digestive system(e.g., salivary glands, esophagus, stomach, liver, gallbladder,pancreas, intestines, rectum, and anus) may be combined; cell lines fromtumors of the respiratory system (e.g., pharynx, larynx, bronchi, lungs,and diaphragm) may be combined; and cell lines derived from tumors ofthe urinary system (e.g., kidneys, ureters, bladder, and urethra) may becombined.

According to various embodiments of the vaccine compositions, thedisclosure provides compositions comprising a combination of cell lines.By way of non-limiting examples, cell line combinations are providedbelow. In each of the following examples, cell line DMS 53, whethermodified or unmodified, is combined with 5 other cancer cell lines inthe associated list.

One or more of the cell lines within each recited combination may bemodified as described herein. In some embodiments, none of the celllines in the combination of cell lines are modified.

(1) NCI-H460, NCIH520, A549, DMS 53, LK-2, and NCI-H23 for the treatmentand/or prevention of NSCLC;

(2) DMS 114, NCI-H196, NCI-H1092, SBC-5, NCI-H510A, NCI-H889, NCI-H1341,NCIH-1876, NCI-H2029, NCI-H841, DMS 53, and NCI-H1694 for the treatmentand/or prevention of SCLC;

(3) DMS 53, PC3, DU-145, LNCAP, NCC-IT, and NTERA-2c1-D1 for thetreatment and/or prevention of prostate cancer;

(4) DMS 53, HCT-15, RKO, HuTu-80, HCT-116, and LS411N for the treatmentand/or prevention of colorectal cancer;

(5) DMS 53, Hs 578T, AU565, CAMA-1, MCF-7, and T-47D for the treatmentand/or prevention of breast cancer including triple negative breastcancer (TNBC);

(6) DMS 53, UM-UC-3, J82, TCCSUP, HT-1376, and SCaBER for the treatmentand/or prevention of bladder cancer;

(7) DMS 53, HSC-4, Detroit 562, KON, HO-1-N-1, and OSC-20 for thetreatment and/or prevention of head and/or neck cancer;

(8) DMS 53, Fu97, MKN74, MKN45, OCUM-1, and MKN1 for the treatmentand/or prevention of stomach cancer;

(9) DMS 53, Hep-G2, JHH-2, JHH-4, JHH-6, Li7, HLF, HuH-6, JHH-5, andHuH-7 for the treatment and/or prevention of liver cancer;

(10) DMS 53, DBTRG-05MG, LN-229, SF-126, GB-1, and KNS-60 for thetreatment and/or prevention of glioblastoma;

(11) DMS 53, TOV-112D, ES-2, TOV-21G, OVTOKO, and MCAS for the treatmentand/or prevention of ovarian cancer;

(12) DMS 53, TE-10, TE-6, TE-4, EC-GI-10, OE33, TE-9, TT, TE-11, OE19,and OE21 for the treatment and/or prevention of esophageal cancer;

(13) DMS 53, A-498, A-704, 769-P, 786-0, ACHN, KMRC-1, KMRC-2, VMRC-RCZ,and VMRC-RCW for the treatment and/or prevention of kidney cancer;

(14) DMS 53, PANC-1, KP-3, KP-4, SUIT-2, and PSN1 for the treatmentand/or prevention of pancreatic cancer;

(15) DMS 53, SNG-M, HEC-1-B, JHUEM-3, RL95-2, MFE-280, MFE-296, TEN,JHUEM-2, AN3-CA, and Ishikawa for the treatment and/or prevention ofendometrial cancer;

(16) DMS 53, RPMI-7951, MeWo, Hs 688(A).T, COLO 829, C32, A-375, Hs294T, Hs 695T, Hs 852T, and A2058 for the treatment and/or prevention ofskin cancer; and

(17) DMS 53, NCI-H28, MSTO-211H, IST-Mes1, ACC-MESO-1, NCI-H2052,NCI-H2452, MPP 89, and IST-Mes2 for the treatment and/or prevention ofmesothelioma.

In some embodiments, the cell lines in the vaccine compositions andmethods described herein include one or more cancer stem cell (CSC) celllines, whether modified or unmodified. One example of a CSC cell line issmall cell lung cancer cell line DMS 53, whether modified or unmodified.CSCs display unique markers that differ depending on the anatomicalorigin of the tumor. Exemplary CSC markers include: prominin-1 (CD133),A2B5, aldehyde dehydrogenase (ALDH1), polycomb protein (Bmi-1),integrin-81 (CD29), hyaluronan receptor (CD44), Thy-1 (CD90), SCFreceptor (CD117), TRA-1-60, nestin, Oct-4, stage-specific embryonicantigen-1 (CD15), GD3 (CD60a), stage-specific embryonic antigen-1(SSEA-1) or (CD15), stage-specific embryonic antigen-4 (SSEA-4),stage-specific embryonic antigen-5 (SSEA-5), and Thomsen-Friedenreichantigen (CD176).

Expression markers that identify cancer cell lines with greaterpotential to have stem cell-like properties differ depending on variousfactors including anatomical origin, organ, or tissue of the primarytumor. Exemplary cancer stem cell markers identified by primary tumorsite are provided in Table 2 and are disclosed across various references(e.g., Gilbert, C A & Ross, AH. J Cell Biochem. (2009); Karsten, U &Goletz, S. SpringerPlus (2013); Zhao, W et al. Cancer Transl Med.(2017)).

Exemplary cell lines expressing one or more markers of cancer stemcell-like properties specific for the anatomical site of the primarytumor from which the cell line was derived are listed in Table 2.Exemplary cancer stem cell lines are provided in Table 3. Expression ofCSC markers was determined using RNA-seq data from the Cancer Cell LineEncyclopedia (CCLE) (retrieved from www.broadinstitute.org/ccle on Nov.23, 2019; Barretina, J et al. Nature. (2012)). The HUGO GeneNomenclature Committee gene symbol was entered into the CCLE search andmRNA expression downloaded for each CSC marker. The expression of a CSCmarker was considered positive if the RNA-seq value (FPKM) was greaterthan 0.

TABLE 2 Exemplary CSC markers by primary tumor anatomical originAnatomical Site CSC Marker CSC Marker of Primary Tumor Common Name GeneSymbol Ovaries Endoglin, CD105 ENG CD117, cKIT KIT CD44 CD44 CD133 PROM1SALL4 SAL4 Nanog NANOG Oct-4 POU5F1 Pancreas ALDH1A1 ALDH1A1 c-Myc MYCEpCAM, TROP1 EPCAM CD44 CD44 Cd133 PROM1 CXCR4 CXCR4 Oct-4 POU5F1 NestinNES BMI-1 BMI1 Skin CD27 CD27 ABCB5 ABCB5 ABCG2 ABCG2 CD166 ALCAM NestinNES CD133 PROM1 CD20 MS4A1 NGFR NGFR Lung ALDH1A1 ALDH1A1 EpCAM, TROP1EPCAM CD90 THY1 CD117, cKIT KIT CD133 PROM1 ABCG2 ABCG2 SOX2 SOX2 LiverNanog NANOG CD90/thy1 THY1 CD133 PROM1 CD13 ANPEP EpCAM, TROP1 EPCAMCD117, cKIT KIT SALL4 SAL4 SOX2 SOX2 Upper Aerodigestive ABCG2 ABCG2Tract (Head and Neck) ALDH1A1 ALDH1A1 Lgr5, GPR49 LGR5 BMI-1 BMI1 CD44CD44 cMET MET Central Nervous System ALDH1A1 ALDH1A1 ABCG2 ABCG2 BMI-1BMI1 CD15 FUT4 CD44 CD44 CD49f, Integrin α6 ITGA6 CD90 THY1 CD133 PROM1CXCR4 CXCR4 CX3CR1 CX3CR1 SOX2 SOX2 c-Myc MYC Musashi-1 MSI1 Nestin NESStomach ALDH1A1 ALDH1A1 ABCB1 ABCB1 ABCG2 ABCG2 CD133 PROM1 CD164 CD164CD15 FUT4 Lgr5, GPR49 LGR5 CD44 CD44 MUC1 MUC1 DLL4 DLL4 Colon (Largeand ALDH1A1 ALDH1A1 Small Intestines) c-myc MYC CD44 CD44 CD133 PROM1Nanog NANOG Musashi-1 MSI1 EpCAM, TROP1 EPCAM Lgr5, GPR49 LGR5 SALL4SAL4 Breast ABCG2 ABCG2 ALDH1A1 ALDH1A1 BMI-1 BMI1 CD133 PROM1 CD44 CD44CD49f, Integrin α6 ITGA6 CD90 THY1 c-myc MYC CXCR1 CXCR1 CXCR4 CXCR4EpCAM, TROP1 EPCAM KLF4 KLF4 MUC1 MUC1 Nanog NANOG SALL4 SAL4 SOX2 SOX2Urinary Tract ALDH1A1 ALDH1A1 CEACAM6, CD66c CEACAM6 Oct4 OCT4 CD44 CD44YAP1 YAP1 Hematopoietic and BMI-1 BMI1 Lymphoid Tissue CD117, c-kit KITCD20 MS4A1 CD27, TNFRSF7 CD27 CD34 CD34 CD38 CD38 CD44 CD44 CD96 CD96GLI-1 GLI1 GLI-2 GLI2 IL-3Rα IL3RA MICL CLEC12A Syndecan-1, CD138 SDC1TIM-3 HAVCR2 Bone ABCG2 ABCG2 CD44 CD44 Endoglin, CD105 ENG Nestin NES

TABLE 3 Cell lines expressing CSC markers Anatomical Site of Cell LineCell Line Cell Line Source Primary Tumor Common Name SourceIdentification Ovaries JHOM-2B RCB RCB1682 OVCAR-3 ATCC HTB-161 OV56ECACC 96020759 JHOS-4 RCB RCB1678 JHOC-5 RCB RCB1520 OVCAR-4 NCI-DTPOVCAR-4 JHOS-2 RCB RCB1521 EFO-21 DSMZ ACC-235 Pancreas CFPAC-1 ATCCCRL-1918 Capan-1 ATCC HTB-79 Panc 02.13 ATCC CRL-2554 SUIT-2 JCRBJCRB1094 Panc 03.27 ATCC CRL-2549 Skin SK-MEL-28 ATCC HTB-72 RVH-421DSMZ ACC-127 Hs 895.T ATCC CRL-7637 Hs 940.T ATCC CRL-7691 SK-MEL-1 ATCCHTB-67 Hs 936.T ATCC CRL-7686 SH-4 ATCC CRL-7724 COLO 800 DSMZ ACC-193UACC-62 NCI-DTP UACC-62 Lung NCI-H2066 ATCC CRL-5917 NCI-H1963 ATCCCRL-5982 NCI-H209 ATCC HTB-172 NCI-H889 ATCC CRL-5817 COR-L47 ECACC92031915 NCI-H1092 ATCC CRL-5855 NCI-H1436 ATCC CRL-5871 COR-L95 ECACC96020733 COR-L279 ECACC 96020724 NCI-H1048 ATCC CRL-5853 NCI-H69 ATCCHTB-119 DMS 53 ATCC CRL-2062 Liver HuH-6 RCB RCB1367 Li7 RCB RCB1941SNU-182 ATCC CRL-2235 JHH-7 JCRB JCRB1031 SK-HEP-1 ATCC HTB-52 Hep3B2.1-7 ATCC HB-8064 Upper Aerodigestive SNU-1066 KCLB 01066 Tract (Headand Neck) SNU-1041 KCLB 01041 SNU-1076 KCLB 01076 BICR 18 ECACC 06051601CAL-33 DSMZ ACC-447 DETROIT 562 ATCC CCL-138 HSC-3 JCRB JCRB0623 HSC-4JCRB JCRB0624 SCC-9 ATCC CRL-1629 YD-8 KCLB 60501 Urinary Tract CAL-29DSMZ ACC-515 KMBC-2 JCRB JCRB1148 253J KCLB 80001 253J-BV KCLB 80002SW780 ATCC CRL-2169 SW1710 DSMZ ACC-426 VM-CUB-1 DSMZ ACC-400 BC-3C DSMZACC-450 Central Nervous KNS-81 JCRB IFO50359 System TM-31 RCB RCB1731NMC-G1 JCRB IFO50467 GB-1 JCRB IFO50489 SNU-201 KCLB 00201 DBTRG-05MGATCC CRL-2020 YKG-1 JCRB JCRB0746 Stomach ECC10 RCB RCB0983 RERF-GC-1BJCRB JCRB1009 TGBC-11-TKB RCB RCB1148 SNU-620 KCLB 00620 GSU RCB RCB2278KE-39 RCB RCB1434 HuG1-N RCB RCB1179 NUGC-4 JCRB JCRB0834 MKN-45 JCRBJCRB0254 SNU-16 ATCC CRL-5974 OCUM-1 JCRB JCRB0192 Colon (Large andC2BBe1 ATCC CRL-2102 Small Intestines) Caco-2 ATCC HTB-37 SNU-1033 KCLB01033 SW1463 ATCC CCL-234 COLO 201 ATCC CCL-224 GP2d ECACC 95090714 LoVoATCC CCL-229 SW403 ATCC CCL-230 CL-14 DSMZ ACC-504 Breast HCC2157 ATCCCRL-2340 HCC38 ATCC CRL-2314 HCC1954 ATCC CRL-2338 HCC1143 ATCC CRL-2321HCC1806 ATCC CRL-2335 HCC1599 ATCC CRL-2331 MDA-MB-415 ATCC HTB-128CAL-51 DSMZ ACC-302 Hematopoietic and KO52 JCRB JCRB0123 Lymphoid TissueSKNO-1 JCRB JCRB1170 Kasumi-1 ATCC CRL-2724 Kasumi-6 ATCC CRL-2775MHH-CALL-3 DSMZ ACC-339 MHH-CALL-2 DSMZ ACC-341 JVM-2 ATCC CRL-3002HNT-34 DSMZ ACC-600 Bone HOS ATCC CRL-1543 OUMS-27 JCRB IFO50488 T1-73ATCC CRL-7943 Hs 870.T ATCC CRL-7606 Hs 706.T ATCC CRL-7447 SJSA-1 ATCCCRL-2098 RD-ES ATCC HTB-166 U2OS ATCC HTB-96 SaOS-2 ATCC HTB-85 SK-ES-1ATCC HTB-86

In certain embodiments, the vaccine compositions comprising acombination of cell lines are capable of stimulating an immune responseand/or preventing cancer and/or treating cancer. The present disclosureprovides compositions and methods of using one or more vaccinecompositions comprising therapeutically effective amounts of cell lines.

The amount (e.g., number) of cells from the various individual celllines in a cocktail or vaccine compositions can be equal (as definedherein) or different. In various embodiments, the number of cells from acell line or from each cell line (in the case where multiple cell linesare administered) in a vaccine composition, is approximately 1.0×10⁶,2.0×10⁶, 3.0×10⁶, 4.0×10⁶, 5.0×10⁶, 6.0×10⁶, 7.0×10⁶, 8×10⁶, 9.0×10⁶,1.0×10⁷, 2.0×10⁷, 3.0×10⁷, 4.0×10⁷, 5.0×10⁷, 6.0×10⁷, 8.0×10⁷, or9.0×10⁷ cells.

The total number of cells administered to a subject, e.g., peradministration site, can range from 1.0×10⁶ to 9.0×10⁷. For example,2.0×10⁶, 3.0×10⁶, 4.0×10⁶, 5.0×10⁶, 6.0×10⁶, 7.0×10⁶, 8×10⁶, 9.0×10⁶,1.0×10⁷, 2.0×10⁷, 3.0×10⁷, 4.0×10⁷, 5.0×10⁷, 6.0×10⁷, 8.0×10⁷, 8.6×10⁷,8.8×10⁷, or 9.0×10⁷ cells are administered.

In certain embodiments, the number of cell lines included in eachadministration of the vaccine composition can range from 1 to 10 celllines. In some embodiments, the number of cells from each cell line arenot equal and different ratios of cell lines are used. For example, ifone cocktail contains 5.0×10⁷ total cells from 3 different cell lines,there could be 3.33×10⁷ cells of one cell line and 8.33×10⁶ of theremaining 2 cell lines.

HLA Diversity

HLA mismatch occurs when the subject's HLA molecules are different fromthose expressed by the cells of the administered vaccine compositions.The process of HLA matching involves characterizing 5 major HLA loci,which include the HLA alleles at three Class I loci HLA-A, -B and -C andtwo class II loci HLA-DRB1 and -DQB1. As every individual expresses twoalleles at each loci, the degree of match or mismatch is calculated on ascale of 10, with 10/10 being a perfect match at all 10 alleles.

The response to mismatched HLA loci is mediated by both innate andadaptive cells of the immune system. Within the cells of the innateimmune system, recognition of mismatches in HLA alleles is mediated tosome extent by monocytes. Without being bound to any theory ormechanism, the sensing of “non-self” by monocytes triggers infiltrationof monocyte-derived DCs, followed by their maturation, resulting inefficient antigen presentation to naïve T cells. Alloantigen-activatedDCs produce increased amounts of IL-12 as compared to DCs activated bymatched syngeneic antigens, and this increased IL-12 production resultsin the skewing of responses to Th1 T cells and increased IFN gammaproduction. HLA mismatch recognition by the adaptive immune system isdriven to some extent by T cells. Without being bound to any theory ormechanism, 1-10% of all circulating T cells are alloreactive and respondto HLA molecules that are not present in self. This is several orders ofmagnitude greater than the frequency of endogenous T cells that arereactive to a conventional foreign antigen. The ability of the immunesystem to recognize these differences in HLA alleles and generate animmune response is a barrier to successful transplantation betweendonors and patients and has been viewed an obstacle in the developmentof cancer vaccines.

As many as 945 different HLA-A and -B alleles can be assigned to one ofthe nine supertypes based on the binding affinity of the HLA molecule toepitope anchor residues. In some embodiments, the vaccine compositionsprovided herein exhibit a heterogeneity of HLA supertypes, e.g.,mixtures of HLA-A supertypes, and HLA-B supertypes. As described herein,various features and criteria may be considered to ensure the desiredheterogeneity of the vaccine composition including, but not limited to,an individual's ethnicity (with regard to both cell donor and subjectreceiving the vaccine). Additional criteria are described herein (e.g.,Example 22). In certain embodiments, a vaccine composition expresses aheterogeneity of HLA supertypes, wherein at least two different HLA-Aand at least two HLA-B supertypes are represented.

In some embodiments, a composition comprising therapeutically effectiveamounts of multiple cell lines are provided to ensure a broad degree ofHLA mismatch on multiple class I and class II HLA molecules between thetumor cell vaccine and the recipient.

In some embodiments, the vaccine composition expresses a heterogeneityof HLA supertypes, wherein the composition expresses a heterogeneity ofmajor histocompatibility complex (MHC) molecules such that two ofHLA-A24, HLA-A03, HLA-A01, and two of HLA-B07, HLA-B08, HLA-B27, andHLA-B44 supertypes are represented. In some embodiments, the vaccinecomposition expresses a heterogeneity HLA supertypes, wherein thecomposition expresses a heterogeneity of MHC molecules and at least theHLA-A24 is represented. In some exemplary embodiments, the compositionexpresses a heterogeneity of MHC molecules such that HLA-A24, HLA-A03,HLA-A01, HLA-B07, HLA-B27, and HLA-B44 supertypes are represented. Inother exemplary embodiments, the composition expresses a geneticheterogeneity of MHC molecules such that HLA-A01, HLA-A03, HLA-B07,HLA-B08, and HLA-B44 supertypes are represented.

Patients display a wide breadth of HLA types that act as markers ofself. A localized inflammatory response that promotes the release ofcytokines, such as IFNγ and IL-2, is initiated upon encountering anon-self cell. In some embodiments, increasing the heterogeneity ofHLA-supertypes within the vaccine cocktail has the potential to augmentthe localized inflammatory response when the vaccine is deliveredconferring an adjuvant effect. As described herein, in some embodiments,increasing the breadth, magnitude, and immunogenicity of tumor reactiveT cells primed by the cancer vaccine composition is accomplished byincluding multiple cell lines chosen to have mismatches in HLA types,chosen, for example, based on expression of certain TAAs. Embodiments ofthe vaccine compositions provided herein enable effective priming of abroad and effective anti-cancer response in the subject with theadditional adjuvant effect generated by the HLA mismatch. Variousembodiments of the cell line combinations in a vaccine compositionexpress the HLA-A supertypes and HLA-B supertypes. Non-limiting examplesare provided in Example 22 herein.

Cell Line Modifications

In certain embodiments, the vaccine compositions comprise cells thathave been modified. Modified cell lines can be clonally derived from asingle modified cell, i.e., genetically homogenous, or derived from agenetically heterogenous population.

Cell lines can be modified to express or increase expression of one ormore immunostimulatory factors, to inhibit or decrease expression of oneor more immunosuppressive factors, and/or to express or increaseexpression of one or more TAAs, including optionally TAAs that have beenmutated in order to present neoepitopes (e.g., designed or enhancedantigens with NSMs) as described herein. Additionally, cell lines can bemodified to express or increase expression of factors that can modulatepathways indirectly, such expression or inhibition of microRNAs.Further, cell lines can be modified to secrete non-endogenous or alteredexosomes.

In addition to modifying cell lines to express a TAA orimmunostimulatory factor, the present disclosure also contemplatesco-administering one or more TAAs (e.g., an isolated TAA or purifiedand/or recombinant TAA) or immunostimulatory factors (e.g.,recombinantly produced therapeutic protein) with the vaccines describedherein.

Thus, in various embodiments, the present disclosure provides a unitdose of a vaccine comprising (i) a first composition comprising atherapeutically effective amount of at least 1, 2, 3, 4, 5 or 6 cancercell lines, wherein the cell line or a combination of the cell linescomprises cells that express at least 5, 10, 15, 20, 25, 30, 35, or 40tumor associated antigens (TAAs) associated with a cancer of a subjectintended to receive said composition, and wherein the composition iscapable of eliciting an immune response specific to the at least 5, 10,15, 20, 25, 30, 35, or 40 TAAs, and (ii) a second composition comprisingone or more isolated TAAs. In other embodiments, the first compositioncomprises a cell line or cell lines that is further modified to (a)express or increase expression of at least 1 immunostimulatory factor,and/or (ii) inhibit or decrease expression of at least 1immunosuppressive factor.

Immunostimulatory Factors

An immunostimulatory protein is one that is membrane bound, secreted, orboth that enhances and/or increases the effectiveness of effector T cellresponses and/or humoral immune responses. Without being bound to anytheory, immunostimulatory factors can potentiate antitumor immunity andincrease cancer vaccine immunogenicity. There are many factors thatpotentiate the immune response. For example, these factors may impactthe antigen-presentation mechanism or the T cell mechanism. Insertion ofthe genes for these factors may enhance the responses to the vaccinecomposition by making the vaccine more immunostimulatory of anti-tumorresponse.

Without being bound to any theory or mechanism, expression ofimmunostimulatory factors by the combination of cell lines included inthe vaccine in the vaccine microenvironment (VME) can modulate multiplefacets of the adaptive immune response. Expression of secreted cytokinessuch as GM-CSF and IL-15 by the cell lines can induce thedifferentiation of monocytes, recruited to the inflammatory environmentof the vaccine delivery site, into dendritic cells (DCs), therebyenriching the pool of antigen presenting cells in the VME. Expression ofcertain cytokines can also mature and activate DCs and Langerhans cells(LCs) already present. Expression of certain cytokines can promote DCsand LCs to prime T cells towards an effector phenotype. DCs thatencounter vaccine cells expressing IL-12 in the VME should primeeffector T cells in the draining lymph node and mount a more efficientanti-tumor response. In addition to enhancing DC maturation, engagementof certain immunostimulatory factors with their receptors on DCs canpromote the priming of T cells with an effector phenotype whilesuppressing the priming of T regulatory cells (Tregs). Engagement ofcertain immunostimulatory factors with their receptors on DCs canpromote migration of DCs and T cell mediated acquired immunity.

In some embodiments of the vaccine compositions provided herein,modifications to express the immunostimulatory factors are not made tocertain cell lines or, in other embodiments, all of the cell linespresent in the vaccine composition.

Provided herein are embodiments of vaccine compositions comprising atherapeutically effective amount of cells from at least one cancer cellline (e.g., GBM cell line), wherein the cell line is modified toincrease production of at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) immunostimulatory factors. In some embodiments, theimmunostimulatory factors are selected from those presented in Table 4.Also provided are exemplary NCBI Gene IDs that can be utilized by askilled artisan to determine the sequences to be introduced in thevaccine compositions of the disclosure. These NCBI Gene IDs areexemplary only.

TABLE 4 Exemplary immunostimulatory factors Factor NCBI Gene Symbol(Gene ID) CCL5 CCL5 (6352) XCL1 XCL1 (6375) Soluble CD40L (CD154) CD40LG(959) Membrane-bound CD40L CD40LG (959) CD36 CD36 (948) GITR TNFRSF18(8784) GM-CSF CSF2 (1437) OX-40 TNFRSF4 (7293) OX-40L TNFSF4 (7292)CD137 (41BB) TNFRSF9 (13604) CD80 (B7-1) CD80 (941) IFNγ IFNG (3458)IL-1β ILI B (3553) IL-2 IL2 (3558) IL-6 IL6 (3569) IL-7 IL7 (3574) IL-9IL9 (3578) IL-12 IL12A (3592) IL12B (3593) IL-15 IL15 (3600) IL-18 IL-18(3606) IL-21 IL21 (59067) IL-23 IL23A (51561) IL12B (3593) TNFα TNF(7124)

In some embodiments, the cell lines of the vaccine composition can bemodified (e.g., genetically modified) to express, overexpress, orincrease the expression of one or more immunostimulatory factorsselected from Table 4. In certain embodiments, the immunostimulatorysequence can be a native human sequence. In some embodiments, theimmunostimulatory sequence can be a genetically engineered sequence. Thegenetically engineered sequence may be modified to increase expressionof the protein through codon optimization, or to modify the cellularlocation of the protein (e.g., through mutation of protease cleavagesites).

For example, at least one (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)of the cancer cell lines in any of the vaccine compositions describedherein may be genetically modified to express or increase expression ofone or more immunostimulatory factors. The immunostimulatory factorsexpressed by the cells within the composition may all be the same, mayall be different, or any combination thereof.

In some embodiments, a vaccine composition comprises a therapeuticallyeffective amount of cells from at least one cancer cell line, whereinthe at least one cell line is modified to express 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more of the immunostimulatory factors of Table 4. In someembodiments, the composition comprises a therapeutically effectiveamount of cells from 2, 3, 4, 5, 6, 7, 8, 9, or 10 cancer cell lines. Insome embodiments, the at least one cell line is modified to increase theproduction of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunostimulatoryfactors of Table 5. In some embodiments, the composition comprises atherapeutically effective amount of cells from 2, 3, 4, 5, 6, 7, 8, 9,or 10 cancer cell lines, and each cell line is modified to increase theproduction of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunostimulatoryfactors of Table 4.

In some embodiments, the composition comprises a therapeuticallyeffective amount of cells from 3 cancer cells lines wherein 1, 2, or all3 of the cell lines have been modified to express or increase expressionof GM-CSF, membrane bound CD40L, and IL-12.

Exemplary combinations of modifications, e.g., where a cell line or celllines have been modified to express or increase expression of more thanone immunostimulatory factor include but are not limited to:GM-CSF+IL-12; CD40L+IL-12; GM-CSF+CD40L; GM-CSF+IL-12+CD40L;GM-CSF+IL-15; CD40L+IL-15; GM-CSF+CD40L; and GM-CSF+IL-15+CD40L, amongother possible combinations.

In certain instances, tumor cells express immunostimulatory factorsincluding the IL-12A (p35 component of IL-12), GM-CSF (kidney celllines), and CD40L (leukemia cell lines). Thus, in some embodiments, celllines may also be modified to increase expression of one or moreimmunostimulatory factors.

In some embodiments, the cell line combination of or cell lines thathave been modified as described herein to express or increase expressionof one or more immunostimulatory factors will express theimmunostimulatory factor or factors at least 2, 3, 4, 5, 6, 7, 8, 9,10-fold or more relative to the same cell line or combination of celllines that have not been modified to express or increase expression ofthe one or more immunostimulatory factors.

Methods to increase immunostimulatory factors in the vaccinecompositions described herein include, but are not limited to,introduction of the nucleotide sequence to be expressed by way of aviral vector or DNA plasmid. The expression or increase in expression ofthe immunostimulatory factors can be stable expression or transientexpression.

In some embodiments, the cancer cells in any of the vaccine compositionsdescribed herein are genetically modified to express CD40 ligand(CD40L). In some embodiments, the CD40L is membrane bound. In someembodiments, the CD40L is not membrane bound. Unless stated otherwise,as used herein CD40L refers to membrane bound CD40L. In someembodiments, the cancer cells in any of the vaccine compositionsdescribed herein are genetically modified to express GM-CSF, membranebound CD40L, GITR, IL-12, and/or IL-15. Exemplary amino acid andnucleotide sequences useful for expression of the one or more of theimmunostimulatory factors provided herein are presented in Table 5.

TABLE 5 Sequences of exemplary immunostimulatory factors Factor SequenceCD154atgatcgaaacatacaaccaaacttctccccgatctgcggccactggactgcccatcagcatgaaaatttttatgtatttacttactgttt(CD40L)ttcttatcacccagatgattgggtcagcactttttgctgtgtatcttcatagaaggttggacaagatagaagatgaaaggaatcttcatga(mem-agattttgtattcatgaaaacgatacagagatgcaacacaggagaaagatccttatccttactgaactgtgaggagattaaaagccagtttbranegaaggctttgtgaaggatataatgttaaacaaagaggagacgaagaaagaaaacagctttgaaatgcctcgtggtgaagaggatagtcaaabound)ttgcggcacatgtcataagtgaggccagcagtaaaacaacatctgtgttacagtgggctgaaaaaggatactacaccatgagcaacaacttggtaaccctggaaaatgggaaacagctgaccgttaaaagacaaggactctattatatctatgcccaagtcaccttctgttccaatcgggaagcttcgagtcaagctccatttatagccagcctctgcctaaagtcccccggtagattcgagagaatcttactcagagctgcaaatacccacagttccgccaaaccttgcgggcaacaatccattcacttgggaggagtatttgaattgcaaccaggtgcttcggtgtttgtcaatgtgactgatccaagccaagtgagccatggcactggcttcacgtcctttggcttactcaaactctga (SEQ ID NO: 1)CD154Atgatcgaaacctacaaccagacctcaccacgaagtgccgccaccggactgcctattagtatgaaaatctttatgtacctgctgacagtgttc(CD40L)ctgatcacccagatgatcggctccgccctgtttgccgtgtacctgcaccggagactggacaagatcgaggatgagcggaacctgcacgaggact(mem-tcgtgtttatgaagaccatccagcggtgcaacacaggcgagagaagcctgtccctgctgaattgtgaggagatcaagagccagttcgagggcbranetttgtgaaggacatcatgctgaacaaggaggagacaaagaaggagaacagcttcgagatgcccagaggcgaggaggattcccagatcgcbound)cgcccacgtgatctctgaggccagctccaagaccacaagcgtgctgcagtgggccgagaagggctactataccatgtctaacaatctggtga(codon-cactggagaacggcaagcagctgaccgtgaagaggcagggcctgtactatatctatgcccaggtgacattctgcagcaatcgcgaggcctctopti-agccaggccccctttatcgccagcctgtgcctgaagagccctggcaggttcgagcgcatcctgctgagagccgccaacacccactcctctgccmized)aagccatgcggacagcagtcaatccacctgggaggcgtgttcgagctgcagccaggagcaagcgtgttcgtgaatgtgactgacccatcacaggtgtctcacggcactggattcacatcatttggactgctgaaactgtga (SEQ ID NO: 2) CD154MIETYNQTSPRSAATGLPISMKIFMYLLIVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQR(CD40L)CNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMPRGEEDSQIAAHVISEASSKTTSVLQ(mem-WAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRbraneAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL (SEQ ID NO: 3)bound) GITRAtggctcagcatggggctatgggggccttcagggctctgtgcggactggctctgctgtgcgctctgtcactggggcagagaccaacaggaggaccaggatgcggacctggcaggctgctgctgggcaccggcacagacgcaaggtgctgtagagtgcacaccacaaggtgctgtcgcgactaccctggcgaggagtgctgttctgagtgggattgcatgtgcgtgcagccagagtttcactgtggcgatccctgctgtaccacatgccgccaccacccatgtccacctggacagggagtgcagtctcagggcaagttcagctttggcttccagtgcatcgactgtgcaagcggcaccttttccggaggacacgagggacactgcaagccctggaccgattgtacacagtttggcttcctgaccgtgttccctggcaacaagacacacaatgccgtgtgcgtgcctggctccccaccagcagagcccctgggctggctgaccgtggtgctgctggccgtggcagcatgcgtgctgctgctgacaagcgcccagctgggactgcacatctggcagctgcggtcccagtgtatgtggccaagagagacccagctgctgctggaggtgcctccatccacagaggacgcccggtcttgccagttccccgaagaggagaggggggaaagaagtgccgaagaaaagggaaggctgggagacctgtgggtg (SEQ ID NO: 4)GITR MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV (SEQ ID NO: 5) GM-CSFatgtggctgcagagcctgctgctcttgggcactgtggcctgcagcatctctgcacccgcccgctcgcccagccccagcacgcagccctgggagcatgtgaatgccatccaggaggcccggcgtctcctgaacctgagtagagacactgctgctgagatgaatgaaacagtagaagtcatctcagaaatgtttgacctccaggagccgacctgcctacagacccgcctggagctgtacaagcagggcctgcggggcagcctcaccaagctcaagggccccttgaccatgatggccagccactacaagcagcactgccctccaaccccggaaacttcctgtgcaacccagattatcacctttgaaagtttcaaagagaacctgaaggactttctgcttgtcatcccctttgactgctgggagccagtccaggagtga (SEQ ID NO: 6)GM-CSFatgtggctgcagtctctgctgctgctgggcaccgtcgcctgttctatttccgcacccgctcgctccccttctccctcaactcagccttgggag(codon-cacgtgaacgccatccaggaggcccggagactgctgaatctgtcccgggacaccgccgccgagatgaacgagacagtggaagtgatctctgagatopti-gttcgatctgcaggagcccacctgcctgcagacaaggctggagctgtacaagcagggcctgcgcggctctctgaccaagctgaagggcccamized)ctgacaatgatggccagccactataagcagcactgcccccctacccccgagacaagctgtgccacccagatcatcacattcgagtcctttaaggagaacctgaaggactttctgctggtcattccatttgattgttgggagcccgtgcaggagtga (SEQ ID NO: 7)GM-CSFMWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE (SEQ ID NO: 8) IL-12atgtgccatcagcaactggttatatcttggttcagtctcgtctttctcgcgtcacccttggtcgctatctgggagcttaaaaaagatgtctacgtcgttgaacttgattggtaccctgatgctccgggggaaatggtggttttgacttgcgatacgccagaagaggatggcataacgtggacactggaccagtcttcagaggttctcgggtctggtaagacactcactatacaggtgaaggagtttggtgacgcaggacaatatacttgccataaaggcggcgaggtgctctcccatagccttctgctccttcataaaaaagaggacgggatatggtcaactgacattctgaaggatcagaaagaaccgaagaacaaaactttcctcagatgcgaggcaaagaactattcaggccgctttacttgctggtggctcactaccatcagcactgacctcactttcagcgtcaagagcagtagaggctcaagtgacccacaaggggttacatgcggggccgctacgttgtctgccgagcgagtcaggggagataataaggaatatgagtatagcgttgaatgccaagaagattcagcctgcccagccgcagaagagagtcttcccatagaagttatggtggacgcagttcataaactgaagtatgagaactatacatcttccttctttattcgcgatatcataaagcctgatcctccgaaaaacttgcaactcaagccgttgaagaatagccgacaggtcgaggtctcttgggagtatccagatacgtggtctaccccgcactcctatttcagtctcaccttctgtgtgcaggtgcaggggaaaagtaagcgggaaaaaaaggaccgggtatttactgataagacctccgctacagtgatttgtagaaagaacgcctctatcagcgtgagagcccaggatagatattattctagtagttggtctgagtgggcctccgtcccttgttccggaagcggagccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggcctatgtgtccagcgcgcagcctcctccttgtggctaccctggtcctcctggaccacctcagtttggcccgaaacctgccggtcgctacacccgatcctggaatgtttccctgccttcatcacagccagaatctgctgagggcagtcagtaacatgctgcagaaggcgcggcaaactctggagttctatccatgtacctccgaggaaattgatcacgaggacattactaaggataaaacaagtacagtagaagcctgtttgcctcttgagctcactaaaaatgagtcatgcttgaacagtcgagagacgagttttatcactaacggttcatgcttggcgtccaggaagacaagctttatgatggcgctctgcctgtcttctatatatgaagaccttaaaatgtaccaagttgagtttaagaccatgaacgccaaacttttgatggaccccaagaggcagatcttccttgatcagaatatgttggcggtgatcgatgaacttatgcaagctttgaacttcaacagtgagacagtgcctcagaaaagttccttggaggaaccggacttctataagaccaagatcaaactgtgcattttgctgcatgcatttagaattcgagccgttacaatcgaccgggtgatgtcatatttgaatgcatcataa (SEQ ID NO: 9) IL-12MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGSGATNFSLLKQAGDVEENPGPMCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 10) IL-15Atgtataggatgcagctgctgtcatgtatcgcactgtccctggcactggtgactaactctaactgggtgaatgtgatctccgacctgaagaagatcgaggacctgatccagtctatgcacatcgatgccaccctgtacacagagtccgacgtgcacccctcttgcaaggtgaccgccatgaagtgtttcctgctggagctgcaggtcatcagcctggagagcggcgacgcatccatccacgataccgtggagaacctgatcatcctggccaacaatagcctgagctccaacggcaatgtgacagagtccggctgcaaggagtgtgaggagctggaggagaagaatatcaaagagttcctgcagtcattcgtccatatcgtccagatgtttatcaataccagt (SEQ ID NO: 11) IL-15MYRMQLLSCIALSLALVTNSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ IDNO: 12) IL-23atgtgccatcagcagctggtcattagttggtttagcctggtctttctggcctcacccctggtcgcaatctgggaactgaagaaggacgtgtacgtggtggagctggactggtatccagatgcaccaggagagatggtggtgctgacctgcgacacacctgaggaggatggcatcacctggacactggatcagagctccgaggtgctgggcagcggcaagaccctgacaatccaggtgaaggagttcggcgacgccggccagtacacatgtcacaagggcggcgaggtgctgtcccactctctgctgctgctgcacaagaaggaggacggcatctggtccacagacatcctgaaggatcagaaggagccaaagaacaagaccttcctgcggtgcgaggccaagaattatagcggccggttcacctgttggtggctgaccacaatctccaccgatctgacattttctgtgaagtctagcaggggctcctctgacccccagggagtgacatgcggagcagccaccctgagcgccgagcgggtgagaggcgataacaaggagtacgagtattctgtggagtgccag gaggacagcgcctgtccagcagcagagg agtccctgcctatcgaagtgatggtggatgccgtgcacaagctgaagtacgagaattatacaagctccttctttatcagggacatcatcaagccagatccccctaagaacctgcagctgaagcccctgaagaatagccgccaggtggaggtgtcctgggagtaccctgacacctggtccacaccacactcttatttcagcctgaccttttgcgtgcaggtgcagggcaagagcaagagggagaagaaggaccgcgtgttcaccgataagacatccgccaccgtgatctgtcggaagaacgccagcatctccgtgagggcccaggatcgctactattctagctcctggagcgagtgggcctccgtgccatgctctggaggaggaggcagcggcggaggaggctccggaggcggcggctctggcggcggcggctccctgggctctcgggccgtgatgctgctgctgctgctgccctggaccgcacagggaagagccgtgccaggaggctctagcccagcatggacacagtgccagcagctgtcccagaagctgtgcaccctggcatggtctgcccaccctctggtgggccacatggacctgagagaggagggcgatgaggagaccacaaacgacgtgcctcacatccagtgcggcgacggctgtgatccacagggcctgagggacaattctcagttctgtctgcagcgcatccaccagggcctgatcttctacgagaagctgctgggcagcgatatctttacaggagagcccagcctgctgcctgactccccagtgggacagctgcacgcctctctgctgggcctgagccagctgctgcagccagagggacaccactgggagacccagcagatcccttctctgagcccatcccagccttggcagcggctgctgctgcggttcaagatcctgagaagcctgcaggcattcgtcgcagtcgcagccagggtgttcgcccacggagccgctactctgagccca (SEQ ID NO: 13) IL-23MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDINYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSGGGGSLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEGDEETTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAVAARVFAHGAATLSP(SEQ ID NO: 14) XCL1atgaggctgctgattctggcactgctgggcatctgctctctgaccgcttacatcgtggaaggagtcggctctgaagtctctgacaagcgcacatgcgtgtctctgaccacacagcgcctgcccgtgagccggatcaagacctacacaatcaccgagggcagcctgagagccgtgatcttcatcacaaagaggggcctgaaggtgtgcgccgaccctcaggcaacctgggtgcgggacgtggtgagaagcatggataggaagtccaacacccggaacaatatgatccagacaaaacccacaggaacccagcagagcactaatacagccgtgacactgaccggg (SEQ ID NO: 15)XCL1MRLLILALLGICSLTAYIVEGVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDWRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTG (SEQ ID NO: 16)

Provided herein is a GITR protein comprising the amino acid sequence ofSEQ ID NO: 4, or a nucleic acid sequence encoding the same, e.g., SEQ IDNO: 5. Provided herein is a vaccine composition comprising one or morecell lines expressing the same.

Provided herein is a GM-CSF protein comprising the amino acid sequenceof SEQ ID NO: 8, or a nucleic acid sequence encoding the same, e.g., SEQID NO: 6 or SEQ ID NO: 7. Provided herein is a vaccine compositioncomprising one or more cell lines expressing the same.

Provided herein is an IL-12 protein comprising the amino acid sequenceof SEQ ID NO: 10, or a nucleic acid sequence encoding the same, e.g.,SEQ ID NO: 9. Provided herein is a vaccine composition comprising one ormore cell lines expressing the same.

Provided herein is an IL-15 protein comprising the amino acid sequenceof SEQ ID NO: 12, or a nucleic acid sequence encoding the same, e.g.,SEQ ID NO: 11. Provided herein is a vaccine composition comprising oneor more cell lines expressing the same.

Provided herein is an IL-23 protein comprising the amino acid sequenceof SEQ ID NO: 14, or a nucleic acid sequence encoding the same, e.g.,SEQ ID NO: 13. Provided herein is a vaccine composition comprising oneor more cell lines expressing the same.

Provided herein is a XCL1 protein comprising the amino acid sequence ofSEQ ID NO: 16, or a nucleic acid sequence encoding the same, e.g., SEQID NO: 15. Provided herein is a vaccine composition comprising one ormore cell lines expressing the same.

In some embodiments, the cancer cells in any of the vaccine compositionsdescribed herein are genetically modified to express one or more ofCD28, B7-H2 (ICOS LG), CD70, CX3CL1, CXCL10(IP10), CXCL9, LFA-1(ITGB2),SELP, ICAM-1, ICOS, CD40, CD27(TNFRSF7), TNFRSF14(HVEM), BTN3A1, BTN3A2,ENTPD1, GZMA, and PERF1.

In some embodiments, vectors contain polynucleotide sequences thatencode immunostimulatory molecules. Exemplary immunostimulatorymolecules may include any of a variety of cytokines. The term “cytokine”as used herein refers to a protein released by one cell population thatacts on one or more other cells as an intercellular mediator. Examplesof such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-beta;platelet-growth factor; transforming growth factors (TGFs) such asTGF-alpha and TGF-beta; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-alpha, beta, and -gamma; colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1 through IL-36,including, IL-1, IL-1alpha, IL-2, IL-3, IL-7, IL-8, IL-9, IL-11, IL-12;IL-15, IL-18, IL-21, IL-23, IL-27, TNF; and other polypeptide factorsincluding LIF and kit ligand (KL). Other immunomodulatory moleculescontemplated for use herein include IRF3, B7.1, B7.2, 4-1BB, CD40 ligand(CD40L), drug-inducible CD40 (iCD40), and the like.

In certain embodiments, polynucleotides encoding the immunostimulatoryfactors are under the control of one or more regulatory elements thatdirect the expression of the coding sequences. In various embodiments,more than one (i.e., 2, 3, or 4) immunostimulatory factors are encodedon one expression vector. In some embodiments, more than one (i.e., 2,3, 4, 5, or 6) immunostimulatory factors are encoded on separateexpression vectors. Lentivirus containing a gene or genes of interest(e.g., GM-CSF, CD40L, or IL-12 and other immunostimulatory molecules asdescribed herein) are produced in various embodiments by transientco-transfection of 293T cells with lentiviral transfer vectors andpackaging plasmids (OriGene) using LipoD293TM In Vitro DNA TransfectionReagent (SignaGen Laboratories).

For lentivirus infection, in some embodiments, cell lines are seeded ina well plate (e.g., 6-well, 12-well) at a density of 1-10×10⁵ cells perwell to achieve 50-80% cell confluency on the day of infection.Eighteen-24 hours after seeding, cells are infected with lentiviruses inthe presence of 10 μg/mL of polybrene. Eighteen-24 hours afterlentivirus infection, cells are detached and transferred to largervessel. After 24-120 hours, medium is removed and replaced with freshmedium supplemented with antibiotics.

Immunosuppressive Factors

An immunosuppressive factor is a protein that is membrane bound,secreted, or both and capable of contributing to defective and reducedcellular responses. Various immunosuppressive factors have beencharacterized in the context of the tumor microenvironment (TME). Inaddition, certain immunosuppressive factors can negatively regulatemigration of LCs and DCs from the dermis to the draining lymph node.

TGFβ1 is a suppressive cytokine that exerts its effects on multipleimmune cell subsets in the periphery as well as in the TME. In the VME,TGFβ1 negatively regulates migration of LCs and DCs from the dermis tothe draining lymph node. Similarly, TGFβ2 is secreted by most tumorcells and exerts immunosuppressive effects similar to TGFβ1.Modification of the vaccine cell lines to reduce TGFβ1 and/or TGFβ2secretion in the VME ensures the vaccine does not further TGFβ-mediatedsuppression of LC or DC migration.

Within the TME, CD47 expression is increased on tumor cells as a mode oftumor escape by preventing macrophage phagocytosis and tumor clearance.DCs also express SIRPα, and ligation of SIRPα on DCs can suppress DCsurvival and activation. Additional immunosuppressive factors in thevaccine that could play a role in the TME and VME include CD276 (B7-H3)and CTLA4. DC contact with a tumor cell expressing CD276 or CTLA4 in theTME dampens DC stimulatory capabilities resulting in decreased T cellpriming, proliferation, and/or promotes proliferation of T cells.Expression of CTLA4 and/or CD276 on the vaccine cell lines could conferthe similar suppressive effects on DCs or LCs in the VME.

In certain embodiments of the vaccine compositions, production of one ormore immunosuppressive factors can be inhibited or decreased in thecells of the cell lines contained therein. In some embodiments,production (i.e., expression) of one or more immunosuppressive factorsis inhibited (i.e., knocked out or completely eliminated) in the cellsof the cell lines contained in the vaccine compositions. In someembodiments, the cell lines can be genetically modified to decrease(i.e., reduce) or inhibit expression of the immunosuppressive factors.In some embodiments, the immunosuppressive factor is excised from thecells completely. In some embodiments, one or more of the cell lines aremodified such that one or more immunosuppressive factor is produced(i.e., expressed) at levels decreased or reduced by at least 5, 10, 15,20, 25, or 30% (i.e., at least 5, 10, 15, 20, 25, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). In someembodiments, the one or more immunosuppressive factors is selected fromthe group presented in Table 6.

Simultaneously, production of one or more immunostimulatory factors,TAAs, and/or neoantigens can be increased in the vaccine compositions asdescribed herein. In some embodiments of the vaccine compositions, inaddition to the partial reduction or complete (e.g., excision and/orexpression at undetectable levels) inhibition of expression of one ormore immunosuppressive factors by the cell, one or more (i.e., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more) of the cell types within the compositionsalso can be genetically modified to increase the immunogenicity of thevaccine, e.g., by ensuring the expression of certain immunostimulatoryfactors, and/or TAAs.

Any combinations of these actions, modifications, and/or factors can beused to generate the vaccine compositions described herein. By way ofnon-limiting example, the combination of decreasing or reducingexpression of immunosuppressive factors by at least 5, 10, 15, 20, 25,or 30% and increasing expression of immunostimulatory factors at least2-fold higher than an unmodified cell line may be effective to improvethe anti-tumor response of tumor cell vaccines. By way of anothernon-limiting example, the combination of reducing immunosuppressivefactors by at least 5, 10, 15, 20, 25, or 30% and modifying cells toexpress certain TAAs in the vaccine composition, may be effective toimprove the anti-tumor response of tumor cell vaccines.

In some embodiments, a cancer vaccine comprises a therapeuticallyeffective amount of cells from at least one cancer cell line, whereinthe cell line is modified to reduce production of at least oneimmunosuppressive factor by the cell line, and wherein the at least oneimmunosuppressive factor is CD47 or CD276. In some embodiments,expression of CTLA4, HLA-E, HLA-G, TGFβ1, and/or TGFβ2 are also reduced.In some embodiments, one or more, or all, cell lines in a vaccinecomposition are modified to inhibit or reduce expression of CD276,TGFβ1, and TGFβ2. In another embodiment, a vaccine composition isprovided comprising three cell lines that have each been modified toinhibit (e.g., knockout) expression of CD276, and reduce expression of(e.g., knockdown) TGFβ1 and TGFβ2.

In some embodiments, a cancer vaccine composition comprises atherapeutically effective amount of cells from a cancer cell linewherein the cell line is modified to reduce expression of at least CD47.In some embodiments, the CD47 is excised from the cells or is producedat levels reduced by at least 5, 10, 15, 20, 25, or 30% (i.e., at least5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100%). In some embodiments, CD47 is excised from thecells or is produced at levels reduced by at least 90%. Production ofadditional immunosuppressive factors can be reduced in one or more celllines. In some embodiments, expression of CD276, CTLA4, HLA-E, HLA-G,TGFβ1, and/or TGFβ2 are also reduced or inhibited. Production of one ormore immunostimulatory factors, TAAs, or neoantigens can be increased inone or more cell lines in these vaccine compositions.

In some embodiments, provided herein is a cancer vaccine compositioncomprising a therapeutically effective amount of cells from a cancercell line wherein the cell line is modified to reduce production of atleast CD276. In some embodiments, the CD276 is excised from the cells oris produced at levels reduced by at least 5, 10, 15, 20, 25, or 30%(i.e., at least 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100%). In some embodiments, CD276 isexcised from the cells or is produced at levels reduced by at least 90%.Production of additional immunosuppressive factors can be reduced in oneor more cell lines. In some embodiments, expression of CD47, CTLA4,HLA-E, HLA-G, TGFβ1, and/or TGFβ2 are also reduced or inhibited.Production of one or more immunostimulatory factors, TAAs, orneoantigens can be increased in one or more cell lines in these vaccinecompositions.

In some embodiments, provided herein is a cancer vaccine compositioncomprising a therapeutically effective amount of cells from a cancercell line wherein the cell line is modified to reduce production of atleast HLA-G. In some embodiments, the HLA-G is excised from the cells oris produced at levels reduced by at least 5, 10, 15, 20, 25, or 30%(i.e., at least 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100%). In some embodiments, HLA-G isexcised from the cells or is produced at levels reduced by at least 90%.Production of additional immunosuppressive factors can be reduced in oneor more cell lines. In some embodiments, expression of CD47, CD276,CTLA4, HLA-E, TGFβ1, and/or TGFβ2 are also reduced or inhibited.Production of one or more immunostimulatory factors, TAAs, orneoantigens can be increased in one or more cell lines in these vaccinecompositions.

In some embodiments, provided herein is a cancer vaccine compositioncomprising a therapeutically effective amount of cells from a cancercell line wherein the cell line is modified to reduce production of atleast CTLA4. In some embodiments, the CTLA4 is excised from the cells oris produced at levels reduced by at least 5, 10, 15, 20, 25, or 30%(i.e., at least 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100%). In some embodiments, CTLA4 isexcised from the cells or is produced at levels reduced by at least 90%.Production of additional immunosuppressive factors can be reduced in oneor more cell lines. In some embodiments, expression of CD47, CD276,HLA-E, TGFβ1, and/or TGFβ2 are also reduced or inhibited. Production ofone or more immunostimulatory factors, TAAs, or neoantigens can beincreased in one or more cell lines in these vaccine compositions.

In some embodiments, provided herein is a cancer vaccine compositioncomprising a therapeutically effective amount of cells from a cancercell line wherein the cell line is modified to reduce production of atleast HLA-E. In some embodiments, the HLA-E is excised from the cells oris produced at levels reduced by at least 5, 10, 15, 20, 25, or 30%(i.e., at least 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100%). In some embodiments, HLA-E isexcised from the cells or is produced at levels reduced by at least 90%.Production of additional immunosuppressive factors can be reduced in oneor more cell lines. In some embodiments, expression of CD47, CD276,CTLA4, TGFβ1, and/or TGFβ2 are also reduced or inhibited. Production ofone or more immunostimulatory factors, TAAs, or neoantigens can beincreased in one or more cell lines in these vaccine compositions.

In some embodiments, provided herein is a cancer vaccine compositioncomprising a therapeutically effective amount of cells from a cancercell line wherein the cell line is modified to reduce production ofTGFβ1, TGFβ2, or both TGFβ1 and TGFβ2. In some embodiments, TGFβ1,TGFβ2, or both TGFβ1 and TGFβ2 is excised from the cells or is producedat levels reduced by at least 5, 10, 15, 20, 25, or 30% (i.e., at least5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100%). In some embodiments of the vaccine composition,TGFβ1, TGFβ2, or both TGFβ1 and TGFβ2 is excised from the cells or isproduced at levels reduced by at least 90%.

In some embodiments, TGFβ1, TGFβ2, or both TGFβ1 and TGFβ2 expression isreduced via a short hairpin RNA (shRNA) delivered to the cells using alentiviral vector. Production of additional immunosuppressive factorscan be reduced. In some embodiments, expression of CD47, CD276, CTLA4,HLA-E, and/or HLA-G are also reduced in one or more cell lines whereTGFβ1, TGFβ2, or both TGFβ1 and TGFβ2 expression is reduced. Productionof one or more immunostimulatory factors, TAAs, or neoantigens can alsobe increased in one or more cell lines in embodiments of these vaccinecompositions.

In some embodiments, the immunosuppressive factor selected for knockdownor knockout may be encoded by multiple native sequence variants.Accordingly, the reduction or inhibition of immunosuppressive factorscan be accomplished using multiple gene editing/knockdown approachesknown to those skilled in the art. As described herein, in someembodiments complete knockout of one or more immunosuppressive factorsmay be less desirable than knockdown. For example, TGFβ1 contributes tothe regulation of the epithelial-mesenchymal transition, so completelack of TGFβ1 (e.g., via knockout) may induce a less immunogenicphenotype in tumor cells.

Table 6 provides exemplary immunosuppressive factors that can beincorporated or modified as described herein, and combinations of thesame. Also provided are exemplary NCBI Gene IDs that can be utilized fora skilled artisan to determine the sequence to be targeted for knockdownstrategies. These NCBI Gene IDs are exemplary only.

TABLE 6 Exemplary immunosuppressive factors Factor NCBI Gene Symbol(Gene ID) B7-H3 (CD276) CD276 (80381) BST2 (CD317) BST2 (684) CD200CD200 (4345) CD39 (ENTPD1) ENTPD1 (953) CD47 CD47 (961) CD73 (NT5E) NT5E(4907) COX-2 PTGS2 (5743) CTLA4 CTLA4 (1493) HLA-E HLA-E (3133) HLA-GHLA-G (3135) IDO (indoleamine IDO1 (3620) 2,3-dioxygenase) IL-10 ID10(3586) PD-L1 (CD274) CD274 (29126) TGFβ1 TGFB1 (7040) TGFβ2 TGFB2 (7042)TGFβ3 TGFB3 (7043) VISTA (VSIR) VSIR (64115) M-CSF CSF1 (1435) B7S1(B7H4) VTCN1 (79679) PTPN2 PTPN2 (5771)

In exemplary embodiments, the production of the following combination ofimmunosuppressive factors is reduced or inhibited in the vaccinecomposition: CD47+TGFβ1, CD47+TGFβ2, or CD47+TGFβ1+TGFβ2. In exemplaryembodiments, the production of the following combination ofimmunosuppressive factors is reduced or inhibited in the vaccinecomposition: CD276+TGFβ1, CD276+TGFβ2, or CD276+TGFβ1+TGFβ2. Inexemplary embodiments, the production of the following combination ofimmunosuppressive factors is reduced or inhibited in the vaccinecomposition: CD47+TGFB1+CD276, CD47+TGFβ2+CD276, orCD47+TGFβ1+TGFβ2+CD276. In exemplary embodiments, the production of thefollowing combination of immunosuppressive factors is reduced orinhibited in the vaccine composition: CD47+TGFβ1+B7-H3,CD47+TGFβ2+CD276, or CD47+TGFβ1+TGFβ2+CD276. In exemplary embodiments,the production of the following combination of immunosuppressive factorsis reduced or inhibited in the vaccine composition:CD47+TGFβ1+CD276+BST2, CD47+TGFβ2+CD276+BST2, orCD47+TGFβ1+TGFβ2+CD276+BST2. In exemplary embodiments, the production ofthe following combination of immunosuppressive factors is reduced orinhibited in the vaccine composition: CD47+TGFβ1+CD276+CTLA4,CD47+TGFβ2+CD276+CTLA4, or CD47+TGFβ1+TGFβ2+CD276+CTLA4. In exemplaryembodiments, the production of the following combination ofimmunosuppressive factors is reduced or inhibited in the vaccinecomposition: CD47+TGFβ1+CD276+CTLA4, CD47+TGFβ2+CD276+CTLA4, orCD47+TGFβ1+TGFβ2+CD276+CTLA4.

In exemplary embodiments, the production of the following combination ofimmunosuppressive factors is reduced or inhibited in the vaccinecomposition: CD47+TGFβ1+CD276+CTLA4, CD47+TGFβ2+CD276+CTLA4, orCD47+TGFβ1+TGFβ2+CD276+CTLA4, CD47+TGFβ2 or TGFβ1+CTLA4, orCD47+TGFβ1+TGFβ2+CD276+HLA-E or CD47+TGFβ1+TGFβ2+CD276+HLA-G, orCD47+TGFβ1+TGFβ2+CD276+HLA-G+CTLA-4, orCD47+TGFβ1+TGFβ2+CD276+HLA-E+CTLA-4.

Those skilled in the art will recognize that in embodiments of thevaccine compositions described herein, at least one (i.e., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more) of the cell lines within the composition hasa knockdown or knockout of at least one immunosuppressive factor (e.g.,one or more of the factors listed in Table 6). The cell lines within thecomposition may have a knockdown or knockout of the sameimmunosuppressive factor, or a different immunosuppressive factor foreach cell line, or of some combination thereof.

Optionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the cell lineswithin the composition may be further genetically modified to have aknockdown or knockout of one or more additional immunosuppressivefactors (e.g., one or more of the factors listed in Table 6). Forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the cell lines withinthe composition may be further genetically modified to have a knockdownor knockout of the same additional immunosuppressive factor, of adifferent additional immunosuppressive factor for each cell line, or ofsome combination thereof.

In some embodiments, provided herein is a cancer vaccine compositioncomprising a therapeutically effective amount of cells from a cancercell line wherein the cell line is modified to reduce production ofSLAMF7, BTLA, EDNRB, TIGIT, KIR2DL1, KIR2DL2, KIR2DL3, TIM3(HAVCR2),LAG3, ADORA2A and ARG1.

At least one of the cells within any of the vaccine compositionsdescribed herein may undergo one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more) genetic modifications in order to achieve the partial orcomplete knockdown of immunosuppressive factor(s) described hereinand/or the expression (or increased expression) of immunostimulatoryfactors described herein, TAAs, and/or neoantigens. In some embodiments,at least one cell line in the vaccine composition undergoes less than 5(i.e., less than 4, less than 3, less than 2, 1, or 0) geneticmodifications. In some embodiments, at least one cell in the vaccinecomposition undergoes no less than 5 genetic modifications.

Numerous methods of reducing or inhibiting expression of one or moreimmunosuppressive factors are known and available to those of ordinaryskill in the art, embodiments of which are described herein.

Cancer cell lines are modified according to some embodiments to inhibitor reduce production of immunosuppressive factors. Provided herein aremethods and techniques for selection of the appropriate technique(s) tobe employed in order to inhibit production of an immunosuppressivefactor and/or to reduce production of an immunosuppressive factor.Partial inhibition or reduction of the expression levels of animmunosuppressive factor may be accomplished using techniques known inthe art.

In some embodiments, the cells of the cancer lines are geneticallyengineered in vitro using recombinant DNA techniques to introduce thegenetic constructs into the cells. These DNA techniques include, but arenot limited to, transduction (e.g., using viral vectors) or transfectionprocedures (e.g., using plasmids, cosmids, yeast artificial chromosomes(YACs), electroporation, liposomes). Any suitable method(s) known in theart to partially (e.g., reduce expression levels by at least 5, 10, 15,20, 25, or 30%) or completely inhibit any immunosuppressive factorproduction by the cells can be employed.

In some embodiments, genome editing is used to inhibit or reduceproduction of an immunosuppressive factor by the cells in the vaccine.Non-limiting examples of genome editing techniques includemeganucleases, zinc finger nucleases (ZFNs), transcriptionactivator-like effector-based nucleases (TALEN), and the CRISPR-Cassystem. In certain embodiments, the reduction of gene expression andsubsequently of biological active protein expression can be achieved byinsertion/deletion of nucleotides via non-homologous end joining (NHEJ)or the insertion of appropriate donor cassettes via homology directedrepair (HDR) that lead to premature stop codons and the expression ofnon-functional proteins or by insertion of nucleotides.

In some embodiments, spontaneous site-specific homologous recombinationtechniques that may or may not include the Cre-Lox and FLP-FRTrecombination systems are used. In some embodiments, methods applyingtransposons that integrate appropriate donor cassettes into genomic DNAwith higher frequency, but with little site/gene-specificity are used incombination with required selection and identification techniques.Non-limiting examples are the piggyBac and Sleeping Beauty transposonsystems that use TTAA and TA nucleotide sequences for integration,respectively.

Furthermore, combinatorial approaches of gene editing methods consistingof meganucleases and transposons can be used.

In certain embodiments, techniques for inhibition or reduction ofimmunosuppressive factor expression may include using antisense orribozyme approaches to reduce or inhibit translation of mRNA transcriptsof an immunosuppressive factor; triple helix approaches to inhibittranscription of the gene of an immunosuppressive factor; or targetedhomologous recombination.

Antisense approaches involve the design of oligonucleotides (either DNAor RNA) that are complementary to mRNA of an immunosuppressive factor.The antisense oligonucleotides bind to the complementary mRNAtranscripts of an immunosuppressive factor and prevent translation.Absolute complementarity may be preferred but is not required. Asequence “complementary” to a portion of an RNA, as referred to herein,means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex. In the case ofdouble-stranded antisense nucleic acids, a single strand of the duplexDNA may be tested, or triplex formation may be assayed. The ability tohybridize depends on both the degree of complementarity and the lengthof the antisense nucleic acid. In some embodiments, oligonucleotidescomplementary to either the 5′ or 3-non-translated, non-coding regionsof an immunosuppressive factor could be used in an antisense approach toinhibit translation of endogenous mRNA of an immunosuppressive factor.In some embodiments, inhibition or reduction of an immunosuppressivefactor is carried out using an antisense oligonucleotide sequence withina short-hairpin RNA.

In some embodiments, lentivirus-mediated shRNA interference is used tosilence the gene expressing the immunosuppressive factor. (See Wei etal., J. Immunother. 2012 35(3)267-275 (2012), incorporated by referenceherein.)

MicroRNAs (miRNA) are stably expressed RNAi hairpins that may also beused for knocking down gene expression. In some embodiments, ribozymemolecules-designed to catalytically cleave mRNA transcripts are used toprevent translation of an immunosuppressive factor mRNA and expression.In certain embodiments, ribozymes that cleave mRNA at site specificrecognition sequences can be used to destroy mRNAs. In some embodiments,the use of hammerhead ribozymes that cleave mRNAs at locations dictatedby flanking regions that form complementary base pairs with the targetmRNA are used. RNA endoribonucleases can also be used.

In some embodiments, endogenous gene expression of an immunosuppressivefactor is reduced by inactivating or “knocking out” the gene or itspromoter, for example, by using targeted homologous recombination. Insome embodiments, endogenous gene expression is reduced by targetingdeoxyribonucleotide sequences complementary to the regulatory region ofthe promoter and/or enhancer genes of an immunosuppressive factor toform triple helical structures that prevent transcription of theimmunosuppressive factor gene in target cells. In some embodiments,promoter activity is inhibited by a nuclease dead version of Cas9(dCas9) and its fusions with KRAB, VP64 and p65 that cannot cleavetarget DNA. The dCas9 molecule retains the ability to bind to target DNAbased on the targeting sequence. This targeting of dCas9 totranscriptional start sites is sufficient to reduce or knockdowntranscription by blocking transcription initiation.

In some embodiments, the activity of an immunosuppressive factor isreduced using a “dominant negative” approach in which genetic constructsthat encode defective immunosuppressive factors are used to diminish theimmunosuppressive activity on neighboring cells.

In some embodiments, the administration of genetic constructs encodingsoluble peptides, proteins, fusion proteins, or antibodies that bind toand “neutralize” intracellularly any other immunosuppressive factors areused. To this end, genetic constructs encoding peptides corresponding todomains of immunosuppressive factor receptors, deletion mutants ofimmunosuppressive factor receptors, or either of these immunosuppressivefactor receptor domains or mutants fused to another polypeptide (e.g.,an IgFc polypeptide) can be utilized. In some embodiments, geneticconstructs encoding anti-idiotypic antibodies or Fab fragments ofanti-idiotypic antibodies that mimic the immunosuppressive factorreceptors and neutralize the immunosuppressive factor are used. Geneticconstructs encoding these immunosuppressive factor receptor peptides,proteins, fusion proteins, anti-idiotypic antibodies or Fabs can beadministered to neutralize the immunosuppressive factor.

Likewise, genetic constructs encoding antibodies that specificallyrecognize one or more epitopes of an immunosuppressive factor, orepitopes of conserved variants of an immunosuppressive factor, orpeptide fragments of an immunosuppressive factor can also be used. Suchantibodies include but are not limited to polyclonal antibodies,monoclonal antibodies (mAbs), humanized or chimeric antibodies, singlechain antibodies, Fab fragments, F(ab′)2 fragments, fragments producedby a Fab expression library, and epitope binding fragments of any of theabove. Any technique(s) known in the art can be used to produce geneticconstructs encoding suitable antibodies.

In some embodiments, the enzymes that cleave an immunosuppressive factorprecursor to the active isoforms are inhibited to block activation ofthe immunosuppressive factor. Transcription or translation of theseenzymes may be blocked by a means known in the art.

In further embodiments, pharmacological inhibitors can be used to reduceenzyme activities including, but not limited to COX-2 and IDO to reducethe amounts of certain immunosuppressive factors.

Tumor Associated Antigens (TAAs)

Vector-based and protein-based vaccine approaches are limited in thenumber of TAAs that can be targeted in a single formulation. Incontrast, embodiments of the allogenic whole cell vaccine platform asdescribed herein allow for the targeting of numerous, diverse TAAs. Thebreadth of responses can be expanded and/or optimized by selectingallogenic cell line(s) that express a range of TAAs and optionallygenetically modifying the cell lines to express additional antigens,including neoantigens or nonsynonymous mutations (NSMs), of interest fora desired therapeutic target (e.g., cancer type).

As used herein, the term “TAA” refers to tumor-associated antigen(s) andcan refer to “wildtype” antigens as naturally expressed from a tumorcell or can optionally refer to a mutant antigen, e.g., a design antigenor designed antigen or enhanced antigen or engineered antigen,comprising one or more mutations such as a neoepitope or one or moreNSMs as described herein.

TAAs are proteins that can be expressed in normal tissue and tumortissue, but the expression of the TAA protein is significantly higher intumor tissue relative to healthy tissue. TAAs may include cancer testisantigens (CTs), which are important for embryonic development butrestricted to expression in male germ cells in healthy adults. CTs areoften expressed in tumor cells.

Neoantigens or neoepitopes are aberrantly mutated genes expressed incancer cells. In many cases, a neoantigen can be considered a TAAbecause it is expressed by tumor tissue and not by normal tissue.Targeting neoepitopes has many advantages since these neoepitopes aretruly tumor specific and not subject to central tolerance in thymus. Acancer vaccine encoding full length TAAs with neoepitopes arising fromnonsynonymous mutations (NSMs) has potential to elicit a more potentimmune response with improved breadth and magnitude.

As used herein, a nonsynonymous mutation (NSM) is a nucleotide mutationthat alters the amino acid sequence of a protein. In some embodiments, amissense mutation is a change in one amino acid in a protein, arisingfrom a point mutation in a single nucleotide. A missense mutation is atype of nonsynonymous substitution in a DNA sequence. Additionalmutations are also contemplated, including but limited to truncations,frameshifts, or any other mutation that change the amino acid sequenceto be different than the native antigen protein.

As described herein, in some embodiments, an antigen is designed by (i)referencing one or more publicly-available databases to identify NSMs ina selected TAA; (ii) identifying NSMs that occur in greater than 2patients; (iii) introducing each NSM identified in step (ii) into therelated TAA sequence; (iv) identifying HLA-A and HLA-Bsupertype-restricted MHC class I epitopes in the TAA that now includesthe NSM; and (v) including the NSMs that create new epitopes (SB and/orWB) or increases peptide-MHC affinity into a final TAA sequence.Exemplary NSMs predicted to create HLA-A and HLA-B supertype-restrictedneoepitopes are provided herein (Table 135).

In some embodiments, an NSM identified in one patient tumor sample isincluded in the designed antigen (i.e., the mutant antigen arising fromthe introduction of the one or more NSMs). In various embodiments, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moreNSMs are introduced into a TAA to generate the designed antigen. In someembodiments, target antigens could have a lower number NSMs and may needto use NSMs occurring only 1 time to reach the targeted homology tonative antigen protein range (94-97%). In other embodiments, targetantigens could have a high number of NSMs occurring at the ≥2 occurrencecut-off and may need to use NSMs occurring 3 times to reach the targetedhomology to native antigen protein range (94-97%). Including a highnumber NSMs in the designed antigen would decrease the homology of thedesigned antigen to the native antigen below the target homology range(94-98%).

In some embodiments, 1, 2, 3, 4, 5 or 6 cell lines of a tumor cellvaccine according to the present disclosure comprise 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more NSMs (andthus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 or more designed antigens) in at least one TAA.

In various embodiments, the sequence homology of the mutant (e.g.,designed antigen) to the native full-length protein is 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% over the full length of the antigen.

In some embodiments, the designed antigen is incorporated into atherapeutic allogenic whole cell cancer vaccine to induceantigen-specific immune responses to the designed TAAs and existingTAAs.

In some embodiments, the vaccine can be comprised of a therapeuticallyeffective amount of at least one cancer cell line, wherein the cell lineor the combination of the cell lines express at least one designed TAA.In other embodiments, the vaccine comprises a therapeutically effectiveamount of at least one cancer cell line, wherein the cell line or thecombination of the cell lines expresses at least 2, 3, 4, 5, 6, 7, 8, 910 or more designed TAAs.

Provided herein are embodiments of vaccine compositions comprising atherapeutically effective amount of cells from at least one cancer cellline, wherein the at least one cancer cell line expresses (eithernatively, or is designed to express) one or more TAAs, neoantigens(including TAAs comprising one or more NSMs), CTs, and/or TAAs. In someembodiments, the cells are transduced with a recombinant lentivectorencoding one or more TAAs, including TAAs comprising one or more NSMs,to be expressed by the cells in the vaccine composition.

In some embodiments, the TAAs, including TAAs comprising one or moreNSMs or neoepitopes, and/or other antigens may endogenously be expressedon the cells selected for inclusion in the vaccine composition. In someembodiments, the cell lines may be modified (e.g., genetically modified)to express selected TAAs, including TAAs comprising one or more NSMs,and/or other antigens (e.g., CTs, TSAs, neoantigens).

Any of the tumor cell vaccine compositions described herein may presentone or more TAAs, including TAAs comprising one or more NSMs orneoepitopes, and induce a broad antitumor response in the subject.Ensuring such a heterogeneous immune response may obviate some issues,such as antigen escape, that are commonly associated with certain cancermonotherapies.

According to various embodiments of the vaccine composition providedherein, at least one cell line of the vaccine composition may bemodified to express one or more neoantigens, e.g., neoantigensimplicated in lung cancer, non-small cell lung cancer (NSCLC), smallcell lung cancer (SCLC), prostate cancer, glioblastoma, colorectalcancer, breast cancer including triple negative breast cancer (TNBC),bladder or urinary tract cancer, squamous cell head and neck cancer(SCCHN), liver hepatocellular (HCC) cancer, kidney or renal cellcarcinoma (RCC) cancer, gastric or stomach cancer, ovarian cancer,esophageal cancer, testicular cancer, pancreatic cancer, central nervoussystem cancers, endometrial cancer, melanoma, and mesothelium cancer. Insome embodiments, one or more of the cell lines expresses an unmutatedportion of a neoantigen protein. In some embodiments, one or more of thecell lines expresses a mutated portion of a neoantigen protein.

In some embodiments, at least one of the cancer cells in any of thevaccine compositions described herein may naturally express, or bemodified to express one or more TAAs, including TAAs comprising one ormore NSMs, CTs, or TSAs/neoantigens. In certain embodiments, more thanone (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the cancer cell linesin the vaccine composition may express, or may be genetically modifiedto express one or more of the TAAs, including TAAs comprising one ormore NSMs, CTs, or TSAs/neoantigens. The TAAs, including TAAs comprisingone or more NSMs, CTs, or TSAs/neoantigens expressed by the cell lineswithin the composition may all be the same, may all be different, or anycombination thereof.

Because the vaccine compositions may contain multiple (i.e., 2, 3, 4, 5,6, 7, 8, 9, 10, or more) cancer cell lines of different types andhistology, a wide range and variety of TAAs, including TAAs comprisingone or more NSMs, and/or neoantigens may be present in the composition(Table 7-23). The number of TAAs that can be targeted using acombination of cell lines (e.g., 5-cell line combination, 6-cell linecombination, 7-cell line combination, 8-cell line combination, 9-cellline combination, or 10-cell line combination) and expression levels ofthe TAAs is higher for the cell line combination compared to individualcell lines in the combination.

In embodiments of the vaccine compositions provided herein, at least one(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the cancer cells inany of the vaccine compositions described herein may express, or bemodified to express one or more TAAs, including TAAs comprising one ormore NSMs or neoepitopes. The TAAs, including TAAs comprising one ormore NSMs, expressed by the cells within the composition may all be thesame, may all be different, or any combination thereof. Table 7 belowlists exemplary non-small cell lung cancer TAAs, and exemplary subsetsof lung cancer TAAs. In some embodiments, the TAAs are specific toNSCLC. In some embodiments, the TAAs are specific to GBM. In otherembodiments, the TAAs are specific to prostate cancer.

In some embodiments, presented herein is a vaccine compositioncomprising a therapeutically effective amount of engineered cells fromleast one cancer cell line, wherein the cell lines or combination ofcell lines express at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40 or more of the TAAs in Tables 7-23. Inother embodiments, the TAAs in Tables 7-23 are modified to include oneor more NSM as described herein.

In some embodiments, a vaccine composition is provided comprising atherapeutically effective amount of engineered cells from at least onecancer cell line, wherein the cell lines express at least 2, 3, 4, 5, 6,7, 8, 9, 10 of the TAAs in Tables 7-23 (or the TAAs in Tables 7-23 thathave been modified to include one or more NSM). As provided herein, invarious embodiments the cell lines express at least 2, 3, 4, 5, 6, 7, 8,9, 10 of the TAAs in Tables 7-23 (or the TAAs in Tables 7-23 that havebeen modified to include one or more NSM) and are optionally modified toexpress or increase expression of one or more immunostimulatory factorsof Table 4, and/or inhibit or decrease expression of one or moreimmunosuppressive factors in Table 6.

TABLE 7 Exemplary TAAs expressed in non-small cell lung cancer TAA NameNCBI Gene Symbol (Gene ID) Survivin BIRC5 (332) CD44 CD44 (960) CD44v6CD44 (960) CEA CEACAM5 (1048) CT83 CT83 (203413) DEPDC1 DEPDC1 (55635)DLL3 DLL3 (10683) NYESO1 CTAG1 (1485) BORIS CTCFL (140690) EGFR EGFR(1956) Her2 ERBB2 (2064) PSMA FOLH1 (2346) KOC1 IGF2BP3 (10643) VEGFRKDR (3791) FLT1 (2321) KIF20A KIF20A (10112) MPHOSPH1 KIF20B (9585) KRASKRAS (3845) LY6K LY6K (54742) MAGE-A1 MAGEA1 (4100) MAGE-A3 MAGEA3(4102) MAGE-A4 MAGEA4 (4103) MAGE-A6 MAGEA6 (4105) Mesothelin MSLN(10232) MUC1 MUC1 (4582) c-Myc MYC (4609) NUF2 NUF2 (83540) FRAME FRAME(23532) CD133 (Prominin-1) PROM1 (8842) PTK7 PTK7 (5754) Securin PTTG1(9232) STEAP1 STEAP1 (26872) hTERT TERT (7015) p53 TP53 (7157) 5T4 TPBG(7162) TTK (CT96) TTK (7272) Brachyury/TBXT T (6862) WT1 WT1 (7490XAGE1B XAGE1B (653067)

TABLE 8 Exemplary TAAs expressed in prostate cancer TAA Name NCBI GeneSymbol (Gene ID) PAP ACP3 (55) Androgen Receptor AR (367) Survivin BIRC5(332) NYESO1 CTAG1B (1485) CXCL12 CXCL12 (6387) CXCR4 CXCR4 (7852) EGFREGFR (1956) Her2 ERBB2 (2064) PSMA FOLH1 (2346) GCNT1 GCNT1 (2650) IDH1IDH1 (3417) FAP FAP (2191) c-KIT/CD117 KIT (3815) PSA KLK3 (354)Galectin 8 LGALS8 (3964) MAGE-A1 MAGEA1 (4100) MAGE-A3 MAGEA3 (4102)MAGE-A4 MAGEA4 (4103) MAGE-C2 MAGEC2 (51438) Midkine MDK (4192) MUC1MUC1 (4582) PDGF-B PDGFB (5155) PDGF-D PDGFD (80310) PDGFRβ PDGFRB(5159) PLAT (T-PA) PLAT (5327) uPA PLAU (5328) uPAR (CD87) PLAUR (5329)CD133 (Prominin-1) PROM1 (8842) PSCA PSCA (8000) SART3 SART3 (9733)Prostein SLC45A3 (85414) CD147 SLC7A11 (23657) SSX2 SSX2 (6757) STEAP1STEAP1 (26872) Brachyury/TBXT T (6862) hTERT TERT (7015) 5T4 TPBG (7162)VEGF-A VEGFA (7422)

TABLE 9 Exemplary TAAs expressed in glioblastoma cancer TAA Name NCBIGene Symbol (Gene ID) AIM2 AIM2 (9447) B4GALNT1 B4GALNT1 (2583) SurvivinBIRC5 (4582) Basigin (BSG) BSG (682) Cyclin B1 CCNB1 (891) CDH5 CDH5(1003) GP39 CHI3L1 (1116) Trp2 DCT (1638) DLL3 DLL3 (10683) DRD2 DRD2(1813) EGFRvIII EGFR (1956) Epha2 EPHA2 (1969) Epha3 EPHA3 (2042) Her2ERBB2 (2064) EZH2 EZH2 (2146) PSMA FOLH1 (2346) FOSL1 FOSL1 (8061) GSK3BGSK3B (2932) IDH1 IDH1 (3417) IDH2 IDH2 (3418) IL13RA2 IL13RA2 (3598)IL4R IL4R (3566) LRP1 LRP1 (4035) KOC1 IGF2BP3 (10643) MAGE-A1 MAGEA1(4100) MAGE-A4 MAGEA4 (4103) MUC1 MUC1 (4582) MUL1 MUL1 (79594) GP100(PM EL) PMEL (6490) PRAME FRAME (23532) hCMV pp65 ABQ23593 (UniProtKB -P06725 (PP65_HCMVA) PROM1 PROM1 (8842) PTHLH PTHLH (4744) SART1 SART1(9092) SART3 SART3 (9733) CD147 SLC7A11 (23657) SOX-2 SOX2 (6657) SOX-11SOX11 (6664) STEAP1 STEAP1 (26872) hTERT TERT (7015) Tenascin-C (TNC)TNC (3371) TYR TYR (7299) Trp1 (TYRP1) TYRP1 (7306) WT1 WT1 (7490) XPO1XPO1 (7514) pp65* ABQ23593 *Viral antigen, no Gene ID is available.Accession number is used instead.

TABLE 10 Exemplary TAAs expressed in ovarian cancer TAA Name NCBI GeneSymbol (Gene ID) OY-TES-1 ACRBP (84519) A-Kinase Anchoring Protein 3AKAP3 (10566) Anti-Mullerian Hormone Receptor AMHR2 (269) Axl ReceptorTyrosine Kinase AXL (558) Survivin BIRC5 (332) Bruton's Tyrosine KinaseBTK (695) CD44 CD44 (960) Cell Cycle Checkpoint Kinase 1 (CHK1) CHEK1(1111) Claudin 6 CLDN6 ((074) NY-ESO-1 CTAG1B (1485) LAGE1 CTAG2 (30848)BORIS CTCFL (140690) Dickkopf-1 DKK1 (22943) DLL4 DLL4 (54567) Her2ERBB2 (2064) HER3 ERBB3 (2065) FOLR1/FBP FOLR1 (2348) GAGE1 GAGE1 (2543)GAGE2 GAGE2A (729447) IGFBP2 IGFBP2 (3485) FSHR FSHR (3969) PLU-1 KDM5B(10765) Luteinizing Hormone Receptor LHCGR (3973) MAGE-A1 MAGEA1 (4100)MAGE-A10 MAGEA10 (4109) MAGE-A4 MAGEA4 (4103) MAGE-A9 MAGEA9 (4108)MAGE-C1 MAGEC1 (9947) Mesothelin MSLN (10232) Muc1 MUC1 (4582) Muc16MUC16 (94025) Glucocorticoid Receptor II NR3C1 (2908) PARP1 PARP1 (142)PIWIL1 PIWIL1 (9271) PIWIL2 PIWIL2 (55124) PIWIL3 PIWIL3 (440822) PIWIL4PIWIL4 (143689) PRAME PRAME (23532) SP17 SPA17 (53340) SPAG-9 SPAG9(9043) STEAP1 STEAP1 (26872) hTERT TERT (7015) WT1 WT1 (7490)

TABLE 11 Exemplary TAAs expressed in colorectal cancer TAA Name NCBIGene Symbol (Gene ID) Survivin BIRC5 (332) B-RAF BRAF (673) CEA CEACAM5(1048) ßHCG CGB3 (1082) NYESO1 CTAG1B (1485) EPCAM EPCAM (4072) EPHreceptor A2 EPHA2 (1969) Her2 ERBB2 (2064) GUCY2C GUCY2C (2984) PSMAFOLH1 (2346) KRAS KRAS (3845) MAGE-A1 MAGEA1 (4100) MAGE-A3 MAGEA3(4102) MAGE-A4 MAGEA4 (4103) MAGE-A6 MAGEA6 (4105) Mesothelin MSLN(10232) MUC1 MUC1 (4582) PRAME PRAME (23532) CD133 PROM1 (8842) RNF43RNF43 (54894) SART3 SART3 (9733) STEAP1 STEAP1 (26872) Brachyury/TBXT T(6862) TROP2 TACSTD2 (4070) hTERT TERT (7015) TOMM34 TOMM34 (10953) 5T4TPBG (7162) WT1 WT1 (7490)

TABLE 12 Exemplary TAAs expressed in breast cancer TAA Name NCBI GeneSymbol (Gene ID) Survivin BIRC5 (332) Cyclin B1 CCNB1 (891) Cadherin-3CDH3 (1001) CEA CEACAM5 (1048) CREB binding protein CREBBP (1387) CS1CSH1 (1442) CT83 CT83 (203413) NYESO1 CTAG1B (1485) BORIS CTCFL (140690)Endoglin ENG (2022) PSMA FOLH1 (2346) FOS like 1 FOSL1 (8061) FOXM1FOXM1 (2305) GPNMB GPNMB (10457) MAGE A1 MAGEA1 (4100) MAGE A3 MAGEA3(4102) MAGE A4 MAGEA4 (4103) MAGE A6 MAGEA6 (4105) Mesothelin MSLN(10232) MMP11 MMP11 (4320) MUC1 MUC1 (4582) PRAME PRAME (23532) CD133PROM1 (8842) PTK7 PTK7 (5754) ROR1 ROR1 (4919) Mammaglobin A SCGB2A2(4250) Syndecan-1 SDC1 (6382) SOX2 SOX2 (6657) SPAG9 SPAG9 (9043) STEAP1STEAP1 (26872) Brachyury/TBXT T (6862) TROP2 TACSTD2 (4070) hTERT TERT(7015) WT1 WT1 (7490) YB-1 YBX1 (4904)

TABLE 13 Exemplary TAAs expressed in bladder cancer Androgen Receptor AR(367) ATG7 ATG7 (10533) AXL Receptor Tyrosine Kinase AXL (558) SurvivinBIRC5 (332) BTK BTK (695) CEACAM1 CEACAM1 (634) CEA CEACAM5 (1048) βHCGCGB3 (1082) NYESO1 CTAG1B (1495) LAGE1 CTAG2 (30848) DEPDC1 DEPDC1(55635) EPH receptor B4 EPHB4 (2050) HER2 ERBB2 (2064) FGFR3 FGFR3(2261) VEGFR FLT3 (2322) PSMA FOLH1 (2346) FOLR1α (FBP) FOLR1 (2348)IGF2BP3 IGF2BP3 (10643) MPHOSPH1 KIF20B (9585) LY6K LY6K (54742) MAGEA1MAGEA1 (4100) MAGEA3 MAGEA3 (4102) MAGEA6 MAGEA6 (4105) MAGEC2 MAGEC2(51438) c-Met MET (4233) MUC1 MUC1 (4582) Nectin-4 NECTIN4 (81607) NUF2NUF2 (83540) RET RET (5979) STEAP1 STEAP1 (26872) TDGF1 (Cripto 1) TDGF1(6997) hTERT TERT (7015) TROP2 TACSTD2 (4070) WEE1 WEE1 (7465) WT1 WT1(7490)

TABLE 14 Exemplary TAAs expressed in head and/or neck cancer TAA NameNCBI Gene Symbol (Gene ID) Survivin BIRC5 (332) BTK BTK (695) cyclin D1CCND1 (595) CDK4 CDK4 (1019) CDK6 CDK6 (1021) P16 CDKN2A (1029) CEACEACAM5 (1048) EGFR EGFR (1956) EPH receptor B4 EPHB4 (2050) Her2 ERBB2(2064) HER3 ERBB3 (2065) FGFR1 FGFR1 (2260) FGFR2 FGFR2 (2263) FGFR3FGFR3 (2261) PSMA FOLH1 (2346) IGF2BP3 IGF2BP3 (10643) IMP3 IMP3 (55272)MPHOSPH1 KIF20B (9585) LY6K LY6K (54742) MAGE-A10 MAGEA10 (4109) MAGE-A3MAGEA3 (4102) MAGE-A4 MAGE-A4 (4103) MAGE-A6 MAGE-A6 (4105) MUC1 MUC1(4582) NUF2 NUF2 (83540) PRAME PRAME (23532) STEAP1 STEAP1 (26872)Brachyury/TBXT T (6862) hTERT TERT (7015) p53 TP53 (7157) HPV16 E6*AVN72023 HPV16 E7* AVN80203 HPV18 E6* ALA62736 HPV18 E7* ABP99745 *Viralantigen, no Gene ID is available; GenBank accession number is provided.

TABLE 15 Exemplary TAAs expressed in gastric cancer TAA Name NCBI GeneSymbol (Gene ID) TEM-8 (ANTXR1) ANTXR1 (84168) Annexin A2 (ANXA2) ANXA2(302) Survivin BIRC5 (332) CCKBR CCKBR (887) Cadherin 17 CDH17 (1015)CDKN2A CDKN2A (1029) CEA CEACAM5 (1048) Claudin 18 CLDN18 (51208) CT83CT83 (203413) EPCAM EPCAM (4072) Her2 ERBB2 (2064) Her3 ERBB3 (2065)PSMA FOLH1 (2346) FOLR1 FOLR1 (2348) FOXM1 FOXM1 (2305) FUT3 FUT3 (2525)Gastrin GAST (2520) KIF20A KIF20A (10112) LY6K LY6K (54742) MAGE-A1MAGEA1 (4100) MAGE-A3 MAGEA3 (4102) MMP9 MMP9 (4318) Mesothelin MSLN(10232) MUC1 MUC1 (4582) MUC3A MUC3A (4584) PRAME PRAME (23532) PTPN11PTPN11 (5781) SART3 SART3 (9733) SATB1 SATB1 (6304) STEAP1 STEAP1(26872) hTERT TERT (7015) 5T4 (TPBG) TPBG (7162) VEGFR1 FLT1 (2321) WEE1WEE1 (7465) WT1 WT1 (7490)

TABLE 16 Exemplary TAAs expressed in liver cancer TAA Name NCBI GeneSymbol (Gene ID) AKR1C3 AKR1C3 (8644) MRP3 (ABCC3) ABCC3 (8714) AFP AFP(174) Annexin A2 (ANXA2) ANXA2 (302) Survivin BIRC5 (4582) Basigin (BSG)BSG (682) CEA CEACAM5 (1048) NYESO1 CTAG1B (1485) DKK-1 DKK1 (22943)SART-2 (DSE) DSE (29940) EpCAM EPCAM (4072) Glypican-3 GPC3 (2719)MAGE-A1 MAGEA1 (4100) MAGE-A3 MAGEA3 (4102) MAGE-A4 MAGEA4 (4103)MAGE-A10 MAGEA10 (4109) MAGE-C1 MAGEC1 (9947) MAGE-C2 MAGEC2 (51438)Midkine (MDK) MDK (4192) MUC-1 MUC1 (4582) PRAME PRAME (23532) SALL-4SALL4 (57167) Spa17 SPA17 (53340) SPHK2 SPHK2 (56848) SSX-2 SSX2 (6757)STAT3 STAT3 (6774) hTERT TERT (7015) HCA661 (TFDP3) TFDP3 (51270) WT1WT1 (7490)

TABLE 17 Exemplary TAAs expressed in esophageal cancer TAA Name NCBIGene Symbol (Gene ID) ABCA1 ABCA1 (19) NYESO1 CTAG1B (1485) LAGE1 CTAG2(30848) DKK1 DKK1 (22943) EGFR EGFR (1956) EpCAM EPCAM (4072) Her2 ERBB2(2065) Her3 ERBB3 (2064) FOLR1 FOLR1 (2348) Gastrin (GAST) GAST (2520)IGF2BP3 IGF2BP3 (10643) IMP3 IMP3 (55272) LY6K LY6K (54742) MAGE-A1MAGEA1 (4100) MAGE-A3 MAGEA3 (4102) MAGE-A4 MAGEA4 (4103) MAGE-A11MAGEA11 (4110) Mesothelin (MSLN) MSLN (10232) NUF2 NUF2 (83540) PRAMEPRAME (23532) PTPN11 PTPN11 (5781) hTERT TERT (7015) TTK TTK (7272)

TABLE 18 Exemplary TAAs expressed in kidney cancer TAA Name NCBI GeneSymbol (Gene ID) apolipoprotein L1 APOL1 (8542) Axl Receptor TyrosineKinase AXL (558) Survivin BIRC5 (332) G250 CA9 (768) cyclin D1 CCND1(595) CXCR4 CXCR4 (7852) EPH receptor B4 EPHB4 (2050) FAP FAP (2191)VEGFR FLT3 (2322) GUCY2C GUCY2C (2984) INTS1 INTS1 (26173) c-KIT/CD117KIT (3815) c-Met MET (4233) MMP7 MMP7 (4316) RAGE1 MOK (5891) Muc1 MUC1(4582) PDGFRα PDGFRA (5156) PDGFRβ PDGFRB (5159) M2PK PKM (5315)perilipin 2 PLIN2 (123) PRAME PRAME (23532) PRUNE2 PRUNE2 (158471) RETRET (5979) RGS5 RGS5 (8490) ROR2 ROR2 (4920) STEAP1 STEAP1 (26872) Tie-1TIE1 (7075) 5T4 TPBG (7162) gp75 TYRP1 (7306)

TABLE 19 Exemplary TAAs expressed in pancreatic cancer TAA Name NCBIGene Symbol (Gene ID) Survivin BIRC5 (332) BTK BTK (695) ConnectiveTissue Growth Factor CCN2 (1490) CEA CEACAM5 (1048) Claudin 18 CLDN18(51208) NYESO1 CTAG1B (1495) CXCR4 CXCR4 (7852) EGFR EGFR (1956) FAP FAP(2191) PSMA FOLH1 (2346) MAGE-A4 MAGEA4 (4103) Perlecan HSPG2 (3339)Mesothelin MSLN (10232) MUC1 MUC1 (4582) Muc16 MUC16 (94025) Mucin 5ACMUC5AC (4586) CD73 NT5E (4907) G17 (gastrin1-17) PBX2 (5089) uPA PLAU(5328) uPAR (CD87) PLAUR (5329) PRAME PRAME (23532) PSCA PSCA (8000)Focal adhesion kinase PTK2 (5747) SSX2 SSX2 (6757) STEAP1 STEAP1 (26872)hTERT TERT (7015) Neurotensin Receptor 1 TFIP11 (24144) WT1 WT1 (7490)

TABLE 20 Exemplary TAAs expressed in endometrial cancer TAA Name NCBIGene Symbol (Gene ID) OY-TES-1 ACRBP (84519) ARMC3 ARMC3 (219681)Survivin BIRC5 (332) BMI1 BMI1 (648) BST2 BST2 (684) BORIS CTCFL(140690) DKK1 DKK1 (22943) DRD2 DRD2 (1813) EpCam EPCAM (4072) EphA2EphA2 (1969) HER2/neu ERBB2 (2064) HER3 ERBB3 (2065 ESR2 ESR2 (2100)MAGE-A3 MAGEA3 (4102) MAGE-A4 MAGEA4 (4103) MAGE-C1 MAGEC1 (9947) MUC-1MUC1 (4582) MUC-16 MUC16 (94025) SPA17 SPA17 (53340) SSX-4 SSX4 (6757)hTERT TERT (7015) HE4 (WFDC2) WFDC2 (10406) WT1 WT1 (7490) XPO1 XPO1(7514)

TABLE 21 Exemplary TAAs expressed in skin cancer TAA Name NCBI GeneSymbol (Gene ID) B4GALNT1 B4GALNT1 (2583) Survivin BIRC5 (332)Endosialin (CD248) CD248 (57124) CDKN2A CDKN2A (1029) CSAG2 CSAG2(102423547) CSPG4 CSPG4 (1464) NYES01 CTAG1B (1485) Trp2 (DCT) DCT(1638) MAGE-A1 MAGEA1 (4100) MAGE-A2 MAGEA2 (4101) MAGE-A3 MAGEA3 (4102)MAGE-A4 MAGEA4 (4103) MAGE-A6 MAGEA6 (4105) MAGE-A10 MAGEA10 (4109) MITFMITF (4286) MART-1 MLANA (2315) NFE2L2 NFE2L2 (4780) PMEL PMEL (6490)PRAME PRAME (23532) NY-MEL-1 RAB38 (23682) NEF S100B (6285) SEMA4DSEMA4D (10507) SSX2 SSX2 (6757) SSX4 SSX4 (6759) ST8SIA1 ST8SIA1 (6489)hTERT TERT (7015) TYR TYR (7299) Trp1 TYRP1 (7306)

TABLE 22 Exemplary TAAs expressed in mesothelial cancer TAA Name NCBIGene Symbol (Gene ID) APEX1 APEX1 (328) CHEK1 CHEK1 (1111) NYESO1 CTAG1B(1485) DHFR DHFR (1719) DKK3 DKK3 (27122) EGFR EGFR (1956) ESR2 ESR2(2100) EZH1 EZH1 (2145) EZH2 EZH2 (2146) MAGE-A1 MAGEA1 (4100) MAGE-A3MAGEA3 (4102) MAGE-A4 MAGEA4 (4103) MCAM MCAM (4162) Mesothelin MSLN(10232) MUC1 MUC1 (4582) PTK2 PTK2 (5747) SSX-2 SSX2 (6757) STAT3 STAT3(6774) THBS2 THBS2 (7058) 5T4 (TPBG) TPBG (7162) WT1 WT1 (7490)

TABLE 23 Exemplary TAAs expressed in small cell lung cancer TAA NameNCBI Gene Symbol (Gene ID) AIM2 AIM2 (9447) AKR1C3 AKR1C3 (8644) ASCL1ASCL1 (429) B4GALNT1 B4GALNT1 (2583) Survivin BIRC5 (332) Cyclin B1CCNB1 (891) CEA CEACAM5 (1048) CKB CKB (1152) DDC DDC (1644) DLL3 DLL3(10863) Enolase 2 ENO2 (2026) Her2 ERBB2 (2064) EZH2 EZH2 (2146)Bombesin GRP (2922) KDM1A KDM1A (23028) MAGE-A1 MAGEA1 (4100) MAGE-A3MAGEA3 (4102) MAGE-A4 MAGA4 (4103) MAGE-A10 MAGEA10 (4109) MDM2 MDM2(4193) MUC1 MUC1 (4582) NCAM-1 NCAM1 (4684) GP100 PMEL (6490) SART-1SART1 (9092) SART-3 SART3 (9733) SFRP1 SFRP1 (6422) SOX-2 SOX2 (6657)SSTR2 SSTR2 (6752) Trp1 (TYRP1) TYRP1 (7306)

In some embodiments of the vaccine compositions provided herein, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more of the cell lines within thecomposition may be genetically modified to express or increaseexpression of the same immunostimulatory factor, TAA, including TAAscomprising one or more NSMs, and/or neoantigen; of a differentimmunostimulatory factor, TAA, and/or neoantigen; or some combinationthereof. In some embodiments, the TAA sequence can be the native,endogenous, human TAA sequence. In some embodiments, the TAA sequencecan be a genetically engineered sequence of the native endogenous, humanTAA sequence. The genetically engineered sequence may be modified toincrease expression of the TAA through codon optimization or thegenetically engineered sequence may be modified to change the cellularlocation of the TAA (e.g., through mutation of protease cleavage sites).

Exemplary NCBI Gene IDs are presented in Table 7-23. As provided herein,these Gene IDs can be used to express (or overexpress) certain TAAs inone or more cell lines of the vaccine compositions of the disclosure.

In various embodiments, one or more of the cell lines in a compositiondescribed herein is modified to express mesothelin (MSLN), CT83(kita-kyushu lung cancer antigen 1) TERT, PSMA, MAGEA1, EGFRvIII, hCMVpp65, TBXT, BORIS, FSHR, MAGEA10, MAGEC2, WT1, FBP, TDGF1, Claudin 18,LY6K, PRAME, HPV16/18 E6/E7, FAP, or mutated versions thereof (Table24). The phrase “or mutated versions thereof” refers to sequences of theaforementioned TAAs, or other TAAs provided herein, that comprise one ormore mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or moresubstitution mutations), including neopepitopes or NSMs, as describedherein. Thus, in various embodiments, one or more of the cell lines in acomposition described herein is modified to express modMesothelin(modMSLN), modTERT, modPSMA, modMAGEA1, modEGFRvIII, modhCMV pp65,modTBXT, modBORIS, modFSHR, modMAGEA10, modMAGEC2, modWT1, modKRAS,modFBP, modTDGF1, modClaudin 18, modLY6K, modFAP, modPRAME, KRAS G12Dmutation, KRAS G12V mutation, and/or modHPV16/18 E6/E7. In otherembodiments, the TAA or “mutated version thereof” may comprise fusionsof 1, 2, or 3 or more of the TAAs or mutated versions provided herein.In some embodiments, the fusions comprises a native or wild-typesequence fused with a mutated TAA. In some embodiments, the individualTAAs in the fusion construct are separated by a cleavage site, such as afurin cleavage site. Thus the present disclosure provides TAA fusionproteins such as CT83-MSLN or modCT83-MSLN, modMAGEA1-EGFRvIII-pp65,modTBXT-modBORIS, modFSHR-modMAGEA10, modTBXT-modMAGEC2, modTBXT-modWT1,modTBXT-modWT1 (KRAS), modWT1-modFBP, modPSMA-modTDGF1,modWT1-modClaudin 18, modPSMA-modLY6K, modFAP-modClaudin 18, andmodPRAME-modTBXT, Sequences for native TAAs can be readily obtained fromthe NCBI database (www.ncbi.nlm.nih.gov/protein). Sequences for theaforementioned TAAs, mutated versions, and fusions are provided in Table24.

TABLE 24 Sequences for MSLN, CT83 and Exemplary Design Antigens TAASequence Mesothelinatggctctgcctaccgcaagacccctgctgggctcctgtgggactcctgctctgggatcactgctgtttctgctgttttcactgggctgggtgcagccttcccgcaccctggcaggagagacaggacaggaggcagcaccactggacggcgtgctggccaacccccctaatatcagctccctgtctcctcggcagctgctgggcttcccatgtgcagaggtgagcggactgtccaccgagagggtgcgcgagctggcagtggccctggcacagaagaacgtgaagctgagcacagagcagctgaggtgcctggcacacaggctgtccgagccaccagaggacctggatgcactgccactggacctgctgctgttcctgaacccagatgccttttccggcccccaggcctgtaccaggttcttttctcgcatcacaaaggccaatgtggatctgctgcccagaggcgcacctgagaggcagagactgctgccagccgccctggcatgctggggcgtgaggggctctctgctgagcgaggcagacgtgcgcgccctgggaggactggcctgtgatctgccaggccgctttgtggcagagagcgccgaggtgctgctgccacggctggtgtcctgccctggcccactggaccaggatcagcaggaggcagcccgggccgccctgcagggcggcggccctccctacggccccccttccacctggtctgtgagcacaatggacgcactgagaggactgctgcctgtgctgggacagccaatcatcaggtctatcccccagggcatcgtggcagcatggaggcagcggagcagccgggaccccagctggcggcagcctgagagaaccatcctgcggcctagattccggagagaggtggagaagacagcctgtccatctggcaagaaggccagagagatcgacgagagcctgatcttttacaagaagtgggagctggaggcctgcgtggacgccgccctgctggctacccagatggacagggtgaatgccatccccttcacctacgagcagctggacgtgctgaagcacaagctggatgagctgtacccacagggctatcccgagtccgtgatccagcacctgggctacctgtttctgaagatgtcccccgaggatatcagaaagtggaacgtgacctctctggagacactgaaggccctgctggaggtcaataagggccacgagatgagccctcaggtggccaccctgatcgaccggttcgtgaagggcagaggccagctggacaaggatacactggataccctgacagccttttaccccggctacctgtgctccctgtctcctgaggagctgtcctctgtgccacccagctccatctgggccgtgcggccacaggacctggatacctgcgacccccggcagctggacgtgctgtaccctaaggccaggctggccttccagaacatgaatggctctgagtatttcgtgaagatccagagctttctgggaggagcacctaccgaggacctgaaggccctgagccagcagaacgtgagcatggacctggccacctttatgaagctgcgcacagatgccgtgctgccactgaccgtggcagaggtgcagaagctgctgggacctcacgtggagggcctgaaggcagaggagaggcacaggccagtgcgggactggattctgcggcagagacaggacgatctggataccctgggactgggactgcagggaggcatcccaaatggaggcagcacatccggctctggcaagccaggctccggagagggctctaccaagggaatgcaggaggccctgagcggcacaccttgcctgctgggacctggacctgtgctgactgtgctggctctgctgctggcatctactctggct (SEQ ID NO: 17) MesothelinMALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGGSTSGSGKPGSGEGSTKGMQEALSGTPCLLGPGPVLTVLALLLASTLA (SEQ ID NO: 18) CT83atgaacttttacctgctgctggcatcctcaatcctgtgcgccctgatcgtgttttggaaataccgacgctttcagagaaatactggcgagatgagcagcaacagcaccgccctggccctggtgcggccctctagctccggcctgatcaactctaatacagacaacaatctggccgtgtacgacctgtctcgggatatcctgaacaatttccctcacagcatcgcccggcagaagagaatcctggtgaacctgagcatggtggagaataagctggtggagctggaacatacactgctgagtaagggctttaggggggcttcaccacatcgcaagtcaaca (SEQ ID NO: 19)CT83 MNFYLLLASSILCALIVFWKYRRFQRNTGEMSSNSTALALVRPSSSGLINSNTDNNLAVYDLSRDILNNFPHSIARQKRILVNLSMVENKLVELEHTLLSKGFRGASPHRKST (SEQ ID NO: 20)CT83-Mesothelinatgaatttctacctgctgctggcatcttcaatcctgtgcgccctgatcgtcttttggaagtatcgccgctttcagaggaacactggcgagatgagcagcaacagcaccgccctggccctggtgcggccttctagctccggcctgatcaactctaatacagacaacaatctggccgtgtatgacctgtcccgggatatcctgaacaatttcccacactctatcgccaggcagaagcgcatcctggtgaacctgagcatggtggagaataagctggtggagctggagcacaccctgctgagcaagggcttccggggagcatccccacacagaaagtctaccggcagcggcgccacaaacttttctctgctgaagcaggcaggcgacgtggaggagaatcctggaccagccctgccaaccgccagacccctgctgggcagctgtggcacacccgccctgggctctctgctgttcctgctgtttagcctgggatgggtgcagccatcaaggaccctggcaggagagacaggacaggaggcagcacccctggatggcgtgctggccaacccccctaatatctctagcctgagcccaagacagctgctgggcttcccatgtgcagaggtgtccggactgtctaccgagagggtgcgcgagctggcagtggccctggcacagaagaatgtgaagctgtctacagagcagctgaggtgcctggcacacagactgagcgagccaccagaggacctggatgcactgcctctggacctgctgctgttcctgaaccccgatgcctttagcggacctcaggcctgcacccggttcttttccagaatcacaaaggccaatgtggatctgctgcctaggggcgcaccagagaggcagagactgctgccagccgccctggcctgctggggcgtgaggggcagcctgctgtccgaggcagacgtgcgcgccctgggaggactggcctgtgatctgccaggccgctttgtggcagagtctgccgaggtgctgctgcctaggctggtgagctgcccaggacctctggaccaggatcagcaggaggcagcccgggccgccctgcagggcggcggccctccatacggccccccttccacctggtccgtgtctacaatggacgcactgagaggactgctgccagtgctgggacagccaatcatcaggagcatcccccagggcatcgtggcagcatggaggcagcggagcagccgggacccctcctggaggcagccagagaggaccatcctgcggccaagattccggagagaggtggagaagacagcatgtccatccggcaagaaggcccgcgagatcgacgagtctctgatcttttacaagaagtgggagctggaggcctgcgtggacgccgccctgctggctacccagatggaccgggtgaacgccatccccttcacctacgagcagctggacgtgctgaagcacaagctggatgagctgtacccccagggctatcctgagtccgtgatccagcacctgggctacctgtttctgaagatgagccccgaggatatccggaagtggaacgtgacctccctggagacactgaaggccctgctggaggtcaataagggccacgagatgagccctcaggtggccaccctgatcgacaggttcgtgaagggccgcggccagctggacaaggatacactggataccctgacagccttttaccctggctacctgtgcagcctgtccccagaggagctgagctccgtgccaccctctagcatctgggccgtgcggccccaggacctggatacctgcgaccctagacagctggatgtgctgtacccaaaggccaggctggccttccagaacatgaatggctctgagtatttcgtgaagatccagagctttctgggaggagcaccaaccgaggacctgaaggccctgtcccagcagaacgtgtctatggacctggccacctttatgaagctgagaacagatgccgtgctgcctctgaccgtggcagaggtgcagaagctgctgggaccacacgtggagggcctgaaggcagaggagaggcacaggcctgtgagggactggattctgcggcagagacaggacgatctggataccctgggactgggactgcagggaggcatccccaatggcggctctacaagcggctccggcaagcctggctctggagagggcagcaccaagggaatgcaggaggccctgagcggcacaccctgtctgctgggacctggacccgtgctgactgtgctggctctgctgctggcttcaaccctggca(SEQ ID NO: 21) CT83-MesothelinMNFYLLLASSILCALIVFWKYRRFQRNTGEMSSNSTALALVRPSSSGLINSNTDNNLAVYDLSRDILNNFPHSIARQKRILVNLSMVENKLVELEHTLLSKGFRGASPHRKSTGSGATNFSLLKQAGDVEENPGPALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLK5ALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGGSTSGSGKPGSGEGSTKGMQEALSGTPCLLGPGPVLTVLALLLASTLA (SEQ ID NO: 22) modTERTatgcctagagcacctagatgtagagctgtgcggagcctgctgcggagccactatagagaagttctgcccctggccaccttcgtgcgtagacttggacctcaaggatggcggctggtgcagagaggcgatcctgctgcttttagagccctggtggcccagtgtctcgtgtgcgttccatgggatgctagacctccaccagctgctcccagcttcagacaggtgtcctgcctgaaagaactggtggccagagtgctgcagcggctgtgtgaaaggggcgccaaaaatgtgctggccttcggctttgccctgctggatgaagctagaggcggacctcctgaggcctttacaacaagcgtgcggagctacctgcctaacaccgtgacagatgccctgagaggatctggcgcttggggactgctgctgagaagagtgggagatgacgtgctggtgcatctgctggcccactgtgctctgtttgtgctggtggctcctagctgcgcctaccaagtttgcggccctctgctgtatcagctgggcgctgctacacaggctagaccacctccacatgccagcggacctagaagaaggctgggctgcgaaagagcctggaaccactctgttagagaagccggcgtgccactgggattgcctgcacctggtgctcggagaagagatggcagcgcctctagatctctgcctctgcctaagaggcccagaagaggcgcagcacctgagcctgagagaacccctatcggccaaggatcttgggcccatcctggcagaacaagaggccctagcgatagaggcttctgcgtggtgtctcctgccagacctgccgaggaagctacatctcttgacggcgccctgagcggcacaagacactctcatccatctgtgggctgccagcaccatgccggacctccatctacaagcagaccacctagaccttgggacaccccttgtcctccagtgtacgccgagacaaagcacttcctgtacagcagcggcgacaaagagcagctgaggcctagcttcctgctgagctttctgaggccaagcctgacaggcgccagacggctgctggaaacaatcttcctgggcagcagaccctggatgcctggcacacttagaaggctgcctagactgccccagcggtactggcaaatgaggcccctgtttctggaactgctgggcaaccacgctcagtgcccttatggcgtgctgctgaaaacccactgtccactgagagccgtggttactccagctgctggcgtgtgtgccagagagaagccacagggatctgtggtggcccctgaggaagaggacaccgatcctagaaggctcgtgcagctgctgaggcagcatagctctccatggcaggtctacggattcgtgcgggcctgtctgcatagactggttccacctggactgtggggctccagacacaacgagcggcggtttctgcggaacaccaagaagttcatcagcctgggaaagcacgccaagctgagcctgcaagagctgacctggaagatgagcgtgtgggattgtgcttggctgcggagaagtcctggcgtgggatgtgttcctgccgccgaacacagactgcgggaagagatcctggccaagttcctgcactggctgatgtccgtgtacgtggtcgaactgctgcggtccctgttctgcgtgaccgagacaaccttccagaagaaccggctgttcttctaccggaagtccgtgtggtccaagctgcagagcatcggcatccggcagcatctgaagagagtgcagctgagagagctgctcgaagccgaagttcggcagcacagaaaagccagactggccctgctgaccagcaggctgagattcatccccaagcacgatggcctgcggcctattgtgaacatggactacgttgtgggcgccagaaccttccaccgggaaaagagagccgagcggctgacctctagagtgaaggccctgtttagcgtgctgaactacgagcgggccagaaggccatctctgctgggagcctttgtgctcggcctggacgatattcatagagcctggcggacattcgtgctgagagtcagagcccaggatagccctcctgagctgtacttcgtgaaggccgatgtgatgggcgcctacaacacaatccctcaggaccggctgaccgagatcattgccagcatcatcaagccccagaacatgtactgtgtgcggagatacgccgtggtgcagaaagccacacatggccacgtgcgcaaggccttcaagagccatgtgtctaccctgaccgacctgcagccttacatgagacagttcgtggcctatctgcaagagacaagccctctgagggacgccgtgatcatcgaacagagcagcagcctgaatgaggccagctccggcctgtttgacgtgttcctcagattcatgtgccaccacgccgtgcggatcagaggcaagagctacatccagtgccagggcattccacagggctccatcctgagcacactgctgtgcagcctgtgctacggcgacatggaaaacaagctgttcgccggcattcggcgcgacggactgcttcttagactggtggacgacttcctgctcgtgacccctcatctgacccacgccaagacctttctgaaaacactcgtgcggggcgtgcccgagtatggctgtgtggtcaatctgagaaagaccgtggtcaacttccccgtcgaggatgaagccctcggcggcacagcttttgtgcagatgcctgctcacggactgttcccttggtgctccctgctgctggacactagaaccctggaagtgcagagcgactacagcagctatgcccggacctctatcagagccagcctgaccttcaaccggggctttaaggccggcagaaacatgcggagaaagctgtttggagtgctgcggctgaagtgccacagcctgttcctcgacctgcaagtgaacagcctgcagaccgtgtgcaccaatatctacaagattctgctgctgcaagcctaccggttccacgcctgtgttctgcagctgcccttccaccagcaagtgtggaagaaccctacattcttcctgcggatcatcagcgacaccgccagcctgtgttacagcatcctgaaggccaagaacgccggcatgtctctgggagctaaaggcgctgcaggacccctgccttttgaagctgttcagtggctgtgtcaccaggcctttctgctgaagctgacccggcacagagtgacatatgtgcccctgctgggctccctgagaacagctcagatgcagctgtccagaaagctgccaggcacaaccctgacagccctggaagctgctgctaaccctgctctgcccagcgacttcaagaccatcctggactgatga(SEQ ID NO: 35) modTERTMPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDEARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLAHCALFVLVAPSCAYQVCGPLLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRDGSASRSLPLPKRPRRGAAPEPERTPIGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLDGALSGTRHSHPSVGCQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSFLRPSLTGARRLLETIFLGSRPWMPGTLRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAVVTPAAGVCAREKPQGSWAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLHRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVWDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSLFCVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELLEAEVRQHRKARLALLTSRLRFIPKHDGLRPIVNMDYVVGARTFHREKRAERLTSRVKALFSVLNYERARRPSLLGAFVLGLDDIHRAWRTFVLRVRAQDSPPELYFVKADVMGAYNTIPQDRLTEIIASIIKPQNMYCVRRYAVVQKATHGHVRKAFKSHVSTLTDLQPYMRQFVAYLQETSPLRDAVIIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYIQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLKTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCSLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRIISDTASLCYSILKAKNAGMSLGAKGAAGPLPFEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQMQLSRKLPGTTLTALEAAANPALPSDFKTILD (SEQ ID NO: 36) modPSMAatgtggaatctgctgcacgagacagatagcgccgtggctaccgttagaaggcccagatggctttgtgctggcgctctggttctggctggcggcttttttctgctgggcttcctgttcggctggttcatcaagagcagcaacgaggccaccaacatcacccctaagcacaacatgaaggcctttctggacgagctgaaggccgagaatatcaagaagttcctgtacaacttcacgcacatccctcacctggccggcaccgagcagaattttcagctggccaagcagatccagagccagtggaaagagttcggcctggactctgtggaactggcccactacgatgtgctgctgagctaccccaacaagacacaccccaactacatcagcatcatcaacgaggacggcaacgagatcttcaacaccagcctgttcgagcctccacctcctggctacgagaacgtgtccgatatcgtgcctccattcagcgctttcagcccacagcggatgcctgagggctacctggtgtacgtgaactacgccagaaccgaggacttcttcaagctggaatgggacatgaagatcagctgcagcggcaagatcgtgatcgcccggtacagaaaggtgttccgcgagaacaaagtgaagaacgcccagctggcaggcgccaaaggcgtgatcctgtatagcgaccccgccgactattttgcccctggcgtgaagtcttaccccgacggctggaattttcctggcggcggagtgcagcggcggaacatccttaatcttaacggcgctggcgaccctctgacacctggctatcctgccaatgagtacgcctacagacacggaattgccgaggctgtgggcctgccttctattcctgtgcaccctgtgcggtactacgacgcccagaaactgctggaaaagatgggcggaagcgcccctcctgactcttcttggagaggctctctgaaggtgccctacaatgtcggcccaggcttcaccggcaacttcagcacccagaaagtgaaaatgcacatccacagcaccaacgaagtgacccggatctacaacgtgatcggcacactgagaggcgccgtggaacccgacaaatacgtgatcctcggcggccacagagacagctgggtgttcggaggaatcgaccctcaatctggcgccgctgtggtgtatgagatcgtgcggtctttcggcaccctgaagaaagaaggatggcggcccagacggaccatcctgtttgcctcttgggacgccgaggaatttggcctgctgggatctacagagtgggccgaagagaacagcagactgctgcaagaaagaggcgtggcctacatcaacgccgacagcagcatcgagggcaactacaccctgcggatcgattgcacccctctgatgtacagcctggtgcacaacctgaccaaagagctgaagtcccctgacgagggctttgagggcaagagcctgtacaagagctggaccaagaagtccccatctcctgagttcagcggcatgcccagaatctctaagctggaaagcggcaacaacttcgaggtgttcttccagcggctgggaatcgcctctggaatcgccagatacaccaagaactgggagacaaacaagttctccggctatcccctgtaccacagcgtgtacgagacatacgagctggtggaaaagttctacgaccccatgttcaagtaccacctgacagtggcccaagtgcgcggaggcatggtgttcgaactggccaatagcatcgtgctgcccttcaactgcagagactacgccgtggtgctgcggaagtacgccgacaagatctacagcatcagcatgaagcacccgcaagagatgaagacctacagcgtgtccttcgactccctgttcttcgccgtgaagaacttcaccaagatcgccagcaagttcagcgagcggctgcaggacttcgacaagagcaaccctatcgtgctgaggatgatgaacgaccagctgatgttcctggaacgggccttcatcaaccctctgggactgcccgacagacccttctacaggcacgtgatctgtgcccctagcagccacaacaaatacgccggcgagagcttccccggcatctacgatgccctgttcgacatcgagagcaacgtgaaccctagcaaggcctggggcgaagtgaagagacagatctacgtggccgcattcacagtgcaggccgctgccgaaacactgtctgaggtggcctgatga (SEQ ID NO: 37) modPSMAMWNLLHETDSAVATVRRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTHIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQRMPEGYLVYVNYARTEDFFKLEWDMKISCSGKIVIARYRKVFRENKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNFPGGGVQRRNILNLNGAGDPLTPGYPANEYAYRHGIAEAVGLPSIPVHPVRYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDKYVILGGHRDSWVFGGIDPQSGAAVVYEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRIDCTPLMYSLVHNLTKELKSPDEGFEGKSLYKSWTKKSPSPEFSGMPRISKLESGNNFEVFFQRLGIASGIARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFNCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFFAVKNFTKIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFINPLGLPDRPFYRHVICAPSSHNKYAGESFPGIYDALFDIESNVNPSKAWGEVKRQIYVAAFTVQAAAETLSEVA (SEQ ID NO: 38) modMAGEA1-EGFRvIII-pp65atgtctctcgaacagagaagcctgcactgcaagcccgaggaagctctggaagctcagcaagaggctctgggccttgtgtgtgttcaggccgctgccagcagcttttctcctctggtgctgggcacactggaagaggtgccaacagccggctctaccgatcctcctcaatctcctcaaggcgccagcgcctttcctaccaccatcaacttcacccggcagagacagcctagcgagggctctagctctcacgaggaaaagggccctagcaccagctgcatcctggaaagcctgttccgggccgtgatcacaaagaaagtggccgacctcgtgggcttcctgctgctgaagtacagagccagagaacccgtgaccaaggccgagatgctggaaagcgtgatcaagaactacaagcactgcttcagcgagatcttcggcaaggccagcgagtctctgcagctcgtgtttggcatcgacgtgaaagaggccgatcctaccggccacagctacgtgttcgtgacatgtctgggcctgagctacgatggcctgctgggcgacaatcagattatgctgaaaaccggcttcctgatcatcgtgctggtcatgatcgccatggaaggctctcacgcccctaaagaggaaatctgggaagaactgagcgtgatggaagtgtacgacggcagagagcatagcgcctacggcgagcctagaaaactgctgacccaggacctggtgcaagagaagtacctcgagtacagacaggtgcccgacagcgaccctgccagatacgaatttctgtggggccctagagcactggccgagacaagctatgtgaaggtgctggaatacgtcatcaaggtgtccgccagagtgtgcttcttcttcccatctctgcgggaagccgctctgcgcgaagaggaagaaggcgtcagaggccggaagagaagaagcctggaagagaaaaagggcaactacgtggtcaccgaccactgcagaggcagaaagcggagaagcgagtctagaggcagacggtgccctgagatgattagcgtgctgggccctatctctggccacgtgctgaaggccgtgttcagcagaggcgatacacctgtgctgccccacgagacaagactgctgcagacaggcatccatgtgcgggtgtcacagccaagcctgatcctggtgtctcagtacacccctgacagcaccccttgtcacagaggcgacaaccagctccaggtgcagcacacctactttaccggcagcgaggtggaaaacgtgtccgtgaacgtgcacaatcccaccggcagatccatctgtcccagccaagagcctatgagcatctacgtgtacgccctgcctctgaagatgctgaacatccccagcatcaatgtgcatcactacccctctgccgccgagcggaaacacagacatctgcctgtggccgatgccgtgattcacgcctctggaaagcagatgtggcaggccagactgacagtgtccggactggcttggaccagacagcagaaccagtggaaagaacccgacgtgtactacacctccgccttcgtgttccccacaaaggacgtggccctgagacacgttgtgtgcgcccatgaactcgtgtgcagcatggaaaacacccgggccaccaagatgcaagtgatcggcgaccagtacgtgaaggtgtacctggaatccttctgcgaggacgtgccaagcggcaagctgttcatgcacgtgaccctgggctccgatgtggaagaggacctgaccatgaccagaaatccccagcctttcatgcggcctcacgagagaaatggcttcaccgtgctgtgccccaagaacatgatcatcaagcccggcaagatcagccacatcatgctggatgtggccttcaccagccacgagcacttcggactgctgtgtcctaagagcatccccggcctgagcatcagcggcaacctgctgatgaatggccagcagatcttcctggaagtgcaggccattcgggaaaccgtggaactgagacagtacgaccctgtggctgccctgttcttcttcgacatcgatctgctgctccagagaggccctcagtacagcgagcacccaacctttaccagccagtacagaatccagggcaagctggaatatcggcacacctgggatagacacgatgagggtgctgcacagggcgacgatgatgtgtggacaagcggcagcgatagcgacgaggaactggtcaccaccgagagaaagacccctagagttacaggcggaggcgcaatggctggcgcttctacatctgccggacgcaagagaaagagcgcctcttctgccaccgcctgtacaagcggcgtgatgacaagaggcaggctgaaagccgagagcacagtggcccctgaggaagatacagacgaggacagcgacaacgagattcacaaccccgccgtgtttacctggcctccttggcaggctggcattctggctagaaacctggtgcctatggtggccacagtgcagggccagaacctgaagtaccaagagttcttctgggacgccaacgacatctaccggatcttcgccgaactggaaggcgtgtggcaaccagccgctcagcccaaaagacgcagacacagacaggacgctctgcccggaccttgtattgccagcacacccaagaaacaccggggctgataa (SEQ ID NO: 39)modMAGEA1-EGFRvIII-pp65MSLEQRSLHCKPEEALEAQQEALGLVCVQAAASSFSPLVLGTLEEVPTAGSTDPPQSPQGASAFPTTINFTRQRQPSEGSSSHEEKGPSTSCILESLFRAVITKKVADLVGFLLLKYRAREPVTKAEMLESVIKNYKHCFSEIFGKASESLQLVFGIDVKEADPTGHSYVFVTCLGLSYDGLLGDNQIMLKTGFLIIVLVMIAMEGSHAPKEEIWEELSVMEVYDGREHSAYGEPRKLLTQDLVQEKYLEYRQVPDSDPARYEFLWGPRALAETSYVKVLEYVIKVSARVCFFFPSLREAALREEEEGVRGRKRRSLEEKKGNYVVTDHCRGRKRRSESRGRRCPEMISVLGPISGHVLKAVFSRGDTPVLPHETRLLQTGIHVRVSQPSLILVSQYTPDSTPCHRGDNQLQVQHTYFTGSEVENVSVNVHNPTGRSICPSQEPMSIYVYALPLKMLNIPSINVHHYPSAAERKHRHLPVADAVIHASGKQMWQARLTVSGLAWTRQQNQWKEPDVYYTSAFVFPTKDVALRHVVCAHELVCSMENTRATKMQVIGDQYVKVYLESFCEDVPSGKLFMHVTLGSDVEEDLTMTRNPQPFMRPHERNGFTVLCPKNMIIKPGKISHIMLDVAFTSHEHFGLLCPKSIPGLSISGNLLMNGQQIFLEVQAIRETVELRQYDPVAALFFFDIDLLLQRGPQYSEHPTFTSQYRIQGKLEYRHTWDRHDEGAAQGDDDVWTSGSDSDEELVTTERKTPRVTGGGAMAGASTSAGRKRKSASSATACTSGVMTRGRLKAESTVAPEEDTDEDSDNEIHNPAVFTWPPWQAGILARNLVPMVATVQGQNLKYQEFFWDANDIYRIFAELEGVWQPAAQPKRRRHRQDALPGPCIASTPKKHRG (SEQ ID NO: 40)modTBXT-modBORISatgtctagccctggaacagagtctgccggcaagagcctgcagtacagagtggaccatctgctgagcgccgtggaaaatgaactgcaggccggaagcgagaagggcgatcctacagagcacgagctgagagtcggcctggaagagtctgagctgtggctgcggttcaaagaactgaccaacgagatgatcgtgaccaagaacggcagacggatgttccccgtgctgaaagtgaacgtgtccggactggaccccaacgccatgtacagctttctgctggacttcgtggtggccgacaaccacagatggaaatacgtgaacggcgagtgggtgccaggcggaaaacctcaactgcaagcccctagctgcgtgtacattcaccctgacagccccaatttcggcgcccactggatgaaggcccctgtgtccttcagcaaagtgaagctgaccaacaagctgaacggcggaggccagatcatgctgaacagcctgcacaaatacgagcccagaatccacatcgtcagagtcggcggaccccagagaatgatcaccagccactgcttccccgagacacagtttatcgccgtgaccgcctaccagaacgaggaaatcaccacactgaagatcaagtacaaccccttcgccaaggccttcctggacgccaaagagcggagcgaccacaaagagatgatcaaagagcccggcgacagccagcagccaggctattctcaatggggatggctgctgccaggcaccagcacattgtgccctccagccaatcctcacagccagtttggaggcgccctgagcctgtctagcacccacagctacgacagataccccacactgcggagccacagaagcagcccctatccttctccttacgctcaccggaacaacagccccacctacagcgataatagccccgcctgtctgagcatgctgcagtcccacgataactggtccagcctgagaatgcctgctcacccttccatgctgcccgtgtctcacaatgcctctccacctaccagcagctctcagtaccctagcctttggagcgtgtccaatggcgccgtgacactgggatctcaggcagccgctgtgtctaatggactgggagcccagttcttcagaggcagccctgctcactacacccctctgacacatcctgtgtctgcccctagcagcagcggcttccctatgtataagggcgctgccgccgctaccgacatcgtggattctcagtatgatgccgccgcacagggacacctgatcgcctcttggacacctgtgtctccaccttccatgagaggcagaaagagaagatccgccgccaccgagatcagcgtgctgagcgagcagttcaccaagatcaaagaattgaagctgatgctcgagaaggggctgaagaaagaagagaaggacggcgtctgccgcgagaagaatcacagaagccctagcgagctggaagcccagagaacatctggcgccttccaggacagcatcctggaagaagaggtggaactggttctggcccctctggaagagagcaagaagtacatcctgacactgcagaccgtgcacttcacctctgaagccgtgcagctccaggacatgagcctgctgtctatccagcagcaagagggcgtgcaggttgtggttcagcaacctggacctggactgctctggctgcaagagggacctagacagtccctgcagcagtgtgtggccatcagcatccagcaagagctgtatagccctcaagagatggaagtgctgcagtttcacgccctcgaagagaacgtgatggtggccatcgaggacagcaagctggctgtgtctctggccgaaacaaccggcctgatcaagctggaagaggaacaagagaagaaccagctgctggccgagaaaacaaaaaagcaactgttcttcgtggaaaccatgagcggcgacgagagaagcgacgagatcgtgctgacagtgtccaacagcaacgtggaagaacaagaggaccagcctaccgcctgtcaggccgatgccgagaaagccaagtttaccaagaaccagagaaagaccaagggcgccaagggcaccttccactgcaacgtgtgcatgttcaccagcagccggatgagcagcttcaactgccacatgaagacccacaccagcgagaagccccatctgtgtcacctgtgcctgaaaaccttccggacagtgacactgctgtggaactatgtgaacacccacacaggcacccggccttacaagtgcaacgactgcaacatggccttcgtgaccagcggagaactcgtgcggcacagaagatacaagcacacccacgagaaacccttcaagtgcagcatgtgcaaatacgcatccatggaagcctccaagctgaagtgccacgtgcgctctcacacaggcgagcaccctttccagtgctgtcagtgtagctacgccagccgggacacctataagctgaagcggcacatgagaacccactctggcgaaaagccctacgagtgccacatctgccacaccagattcacccagagcggcaccatgaagattcacatcctgcagaaacacggcaagaacgtgcccaagtaccagtgtcctcactgcgccaccattatcgccagaaagtccgacctgcgggtgcacatgaggaatctgcacgcctattctgccgccgagctgaaatgcagatactgcagcgccgtgttccacaagagatacgccctgatccagcaccagaaaacccacaagaacgagaagcggtttaagtgcaagcactgcagctacgcctgcaagcaagagcgccacatgatcgcccacatccacacacacaccggggagaagccttttacctgcctgagctgcaacaagtgcttccggcagaaacagctgctcaacgcccacttcagaaagtaccacgacgccaacttcatccccaccgtgtacaagtgctccaagtgcggcaagggcttcagccggtggatcaatctgcaccggcacctggaaaagtgcgagtctggcgaagccaagtctgccgcctctggcaagggcagaagaacccggaagagaaagcagaccatcctgaaagaggccaccaagagccagaaagaagccgccaagcgctggaaagaggctgccaacggcgacgaagctgctgccgaagaagccagcacaacaaagggcgaacagttccccgaagagatgttccctgtggcctgcagagaaaccacagccagagtgaagcaagaggtcgaccagggcgtgacctgcgagatgctgctgaacaccatggacaagtgatga (SEQ ID NO: 41)modTBXT-modBORISMSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTEHELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVVADNHRWKYVNGEWVPGGKPQLQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITTLKIKYNPFAKAFLDAKERSDHKEMIKEPGDSQQPGYSQWGWLLPGTSTLCPPANPHSQFGGALSLSSTHSYDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLRMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTLGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGFPMYKGAAAATDIVDSQYDAAAQGHLIASWTPVSPPSMRGRKRRSAATEISVLSEQFTKIKELKLMLEKGLKKEEKDGVCREKNHRSPSELEAQRTSGAFQDSILEEEVELVLAPLEESKKYILTLQTVHFTSEAVQLQDMSLLSIQQQEGVQVVVQQPGPGLLWLQEGPRQSLQQCVAISIQQELYSPQEMEVLQFHALEENVMVAIEDSKLAVSLAETTGLIKLEEEQEKNQLLAEKTKKQLFFVETMSGDERSDEIVLTVSNSNVEEQEDQPTACQADAEKAKFTKNQRKTKGAKGTFHCNVCMFTSSRMSSFNCHMKTHTSEKPHLCHLCLKTFRTVTLLWNYVNTHTGTRPYKCNDCNMAFVTSGELVRHRRYKHTHEKPFKCSMCKYASMEASKLKCHVRSHTGEHPFQCCQCSYASRDTYKLKRHMRTHSGEKPYECHICHTRFTQSGTMKIHILQKHGKNVPKYQCPHCATIIARKSDLRVHMRNLHAYSAAELKCRYCSAVFHKRYALIQHQKTHKNEKRFKCKHCSYACKQERHMIAHIHTHTGEKPFTCLSCNKCFRQKQLLNAHFRKYHDANFIPTVYKCSKCGKGFSRWINLHRHLEKCESGEAKSAASGKGRRTRKRKQTILKEATKSQKEAAKRWKEAANGDEAAAEEASTTKGEQFPEEMFPVACRETTARVKQEVDQGVTCEMLLNTMDK (SEQ ID NO: 42) modFSHR-modMAGEA10atggctctgctgctggtttctctgctggccctgctgtctctcggctctggatgtcaccacagaatctgccactgcagcaaccgggtgttcctgtgccagaaaagcaaagtgaccgagatcctgagcgacctgcagcggaatgccatcgagctgagattcgtgctgaccaagctgcaagtgatccagaagggcgccttcagcggcttcggcgacctggaaaagatcgagatcagccagaacaacgtgctggaagtgatcgaggcccacgtgttcagcaacctgcctaagctgcacgagatcagaatcgagaaggccaacaacctgctgtacatcaaccccgaggccttccagaacttccccaacctgcagtacctgctgatctccaacaccggcatcaaacatctgcccgacgtgcacaagatccacagcctgcagaaggtgctgctggacatccaggacaacatcaacatccacacaatcgagcggaactacttcctgggcctgagcttcgagagcgtgatcctgtggctgaacaagaacggcatccaagagatccacaactgcgccttcaatggcacccagctggacgagctgaacctgtccgacaacaacaatctggaagaactgcccaacgacgtgttccacagagccagcggacctgtgatcctggacatcagcagaaccagaatccactctctgcccagctacggcctggaaaacctgaagaagctgcgggccagaagcacctacaatctgaaaaagctgcctacgctggaaaccctggtggccctgatggaagccagcctgacataccctagccactgctgcgcctttgccaactggcggagacagatctctgagctgcaccccatctgcaacaagagcatcctgcggcaagaggtggactacatgacacaggccagaggccagagattcagcctggccgaggataacgagagcagctacagcagaggcttcgacatgacctacaccgagttcgactacgacctgtgcaacaaggtggtggacgtgacatgcagccccaagcctgatgccttcaatccctgcgaggacatcatgggctacaacatcctgagagtgctgatctggttcatcagcatcctggccatcaccgagaacatcatcgtgctggtcatcctgaccaccagccagtacaagctgaccgtgcctatgttcctgatgtgcaacctggccttcgccgatctgtgcatcggcatctacctgctgctgatcgccagcgtggacattcacaccaagagccagtaccacaactacgccatcgactggcagacaggcgccggatgtgatgccgccggattctttacagtgttcgccagcgagctgtccgtgtacaccctgacagctatcaccctggaacggtggcacaccatcacacacgctatgcagctggactgcaaagtgcacctgagacacagcgcctccgtgatggttatgggctggatcttcgccttcgctgccgctctgttccccatctttggcatcagctcctacatgaaggtgtccatctatctgcccatggacatcgacagccctctgagccagctgtacgtgatgagtctgctggtgctgaatgtgctggcctttgtggtcatctgcggctgctacatctatatctacctgacagtgcggaaccccaacatcgtgtccagctccagcgacacccggatcgctaagagaatggccatgctgatcttcaccgactttctgtgcatggcccctatcagcctgttcgccattagcgctagcctgaaggtgcccctgatcaccgtgtccaaggccaagattctgctggtcctgttctaccccatcaacagctgcgccaatcctttcctgtacgccatcttcaccaagaacttcaggcggaacttcttcatcctgctgagcaagcggggctgttacaagatgcaggcccagatctaccggaccgagacactgtccaccgtgcacaacacacaccccagaaacggccactgtagcagcgcccctagagtgacaaatggctccacctacatcctggtgccactgagccatctggcccagaacagaggccggaagagaagaagccccagggctcccaagagacagagatgcatgcccgaagaggacctgcagagccagagcgaaacacagggactcgaaggtgctcaggctcctctggccgtggaagaagatgccagcagctctaccagcacctccagcagcttccctagcagctttccattcagctcctctagctctagcagcagctgttaccctctgatccccagcacacccgagaaggtgttcgccgacgacgagacacctaatccactgcagtctgcccagatcgcctgcagcagtacactggtggttgctagcctgcctctggaccagtctgatgagggaagcagcagccagaaagaggaaagccctagcacactccaggtgctgcccgatagcgagagcctgcctagaagcgagatctacaagaaaatgaccgacctggtgcagttcctcctgttcaagtaccagatgaaggaacccatcaccaaggccgaaatcctggaaagcgtgatcagaaactacgaggaccactttccactgctgttcagcgaggccagcgagtgcatgctgctcgtgtttagcatcgacgtgaagaaggtggaccccaccggccacagctttgtgctggttacaagcctgggactgacctacgacggcatgctgtccgatgtgcagagcatgcctaagaccggcatcctgatcctgattctgagcatcgtgttcatcgagggctactgcacccctgaggaagtgatttgggaagccctgaacatgatgggcctgtacgatggcatggaacacctgatctacggcgagcccagaaaactgctgacccaggactgggtgcaagagaactacctggaataccggcagatgcccggcagcgatcctgccagatatgagtttctgtggggccctagagcacatgccgagatccggaagatgagcctgctgaagttcctggccaaagtgaacggcagcgacccaatcagcttcccactttggtacgaagaggccctgaaggacgaggaagagagagcccaggatagaatcgccaccaccgacgacacaacagccatggcctctgcctcttctagcgccaccggcagctttagctaccccgagtgataa (SEQ ID NO: 43) modFSHR-modMAGEA10MALLLVSLLALLSLGSGCHHRICHCSNRVFLCQKSKVTEILSDLQRNAIELRFVLTKLQVIQKGAFSGFGDLEKIEISQNNVLEVIEAHVFSNLPKLHEIRIEKANNLLYINPEAFQNFPNLQYLLISNTGIKHLPDVHKIHSLQKVLLDIQDNINIHTIERNYFLGLSFESVILWLNKNGIQEIHNCAFNGTQLDELNLSDNNNLEELPNDVFHRASGPVILDISRTRIHSLPSYGLENLKKLRARSTYNLKKLPTLETLVALMEASLTYPSHCCAFANWRRQISELHPICNKSILRQEVDYMTQARGQRFSLAEDNESSYSRGFDMTYTEFDYDLCNKVVDVTCSPKPDAFNPCEDIMGYNILRVLIWFISILAITENIIVLVILTTSQYKLTVPMFLMCNLAFADLCIGIYLLLIASVDIHTKSQYHNYAIDWQTGAGCDAAGFFTVFASELSVYTLTAITLERWHTITHAMQLDCKVHLRHSASVMVMGWIFAFAAALFPIFGISSYMKVSIYLPMDIDSPLSQLYVMSLLVLNVLAFVVICGCYIYIYLTVRNPNIVSSSSDTRIAKRMAMLIFTDFLCMAPISLFAISASLKVPLITVSKAKILLVLFYPINSCANPFLYAIFTKNFRRNFFILLSKRGCYKMQAQIYRTETLSTVHNTHPRNGHCSSAPRVTNGSTYILVPLSHLAQNRGRKRRSPRAPKRQRCMPEEDLQSQSETQGLEGAQAPLAVEEDASSSTSTSSSFPSSFPFSSSSSSSSCYPLIPSTPEKVFADDETPNPLQSAQIACSSTLVVASLPLDQSDEGSSSQKEESPSTLQVLPDSESLPRSEIYKKMTDLVQFLLFKYQMKEPITKAEILESVIRNYEDHFPLLFSEASECMLLVFSIDVKKVDPTGHSFVLVTSLGLTYDGMLSDVQSMPKTGILILILSIVFIEGYCTPEEVIWEALNMMGLYDGMEHLIYGEPRKLLTQDWVQENYLEYRQMPGSDPARYEFLWGPRAHAEIRKMSLLKFLAKVNGSDPISFPLWYEEALKDEEERAQDRIATTDDTTAMASASSSATGSFSYPE (SEQ IDNO: 44) modTBXT-modMAGEC2atggctctgctgctggtttctctgctggccctgctgtctctcggctctggatgtcaccacagaatctgccactgcagcaaccgggtgttcctgtgccagaaaagcaaagtgaccgagatcctgagcgacctgcagcggaatgccatcgagctgagattcgtgctgaccaagctgcaagtgatccagaagggcgccttcagcggcttcggcgacctggaaaagatcgagatcagccagaacaacgtgctggaagtgatcgaggcccacgtgttcagcaacctgcctaagctgcacgagatcagaatcgagaaggccaacaacctgctgtacatcaaccccgaggccttccagaacttccccaacctgcagtacctgctgatctccaacaccggcatcaaacatctgcccgacgtgcacaagatccacagcctgcagaaggtgctgctggacatccaggacaacatcaacatccacacaatcgagcggaactacttcctgggcctgagcttcgagagcgtgatcctgtggctgaacaagaacggcatccaagagatccacaactgcgccttcaatggcacccagctggacgagctgaacctgtccgacaacaacaatctggaagaactgcccaacgacgtgttccacagagccagcggacctgtgatcctggacatcagcagaaccagaatccactctctgcccagctacggcctggaaaacctgaagaagctgcgggccagaagcacctacaatctgaaaaagctgcctacgctggaaaccctggtggccctgatggaagccagcctgacataccctagccactgctgcgcctttgccaactggcggagacagatctctgagctgcaccccatctgcaacaagagcatcctgcggcaagaggtggactacatgacacaggccagaggccagagattcagcctggccgaggataacgagagcagctacagcagaggcttcgacatgacctacaccgagttcgactacgacctgtgcaacaaggtggtggacgtgacatgcagccccaagcctgatgccttcaatccctgcgaggacatcatgggctacaacatcctgagagtgctgatctggttcatcagcatcctggccatcaccgagaacatcatcgtgctggtcatcctgaccaccagccagtacaagctgaccgtgcctatgttcctgatgtgcaacctggccttcgccgatctgtgcatcggcatctacctgctgctgatcgccagcgtggacattcacaccaagagccagtaccacaactacgccatcgactggcagacaggcgccggatgtgatgccgccggattctttacagtgttcgccagcgagctgtccgtgtacaccctgacagctatcaccctggaacggtggcacaccatcacacacgctatgcagctggactgcaaagtgcacctgagacacagcgcctccgtgatggttatgggctggatcttcgccttcgctgccgctctgttccccatctttggcatcagctcctacatgaaggtgtccatctatctgcccatggacatcgacagccctctgagccagctgtacgtgatgagtctgctggtgctgaatgtgctggcctttgtggtcatctgcggctgctacatctatatctacctgacagtgcggaaccccaacatcgtgtccagctccagcgacacccggatcgctaagagaatggccatgctgatcttcaccgactttctgtgcatggcccctatcagcctgttcgccattagcgctagcctgaaggtgcccctgatcaccgtgtccaaggccaagattctgctggtcctgttctaccccatcaacagctgcgccaatcctttcctgtacgccatcttcaccaagaacttcaggcggaacttcttcatcctgctgagcaagcggggctgttacaagatgcaggcccagatctaccggaccgagacactgtccaccgtgcacaacacacaccccagaaacggccactgtagcagcgcccctagagtgacaaatggctccacctacatcctggtgccactgagccatctggcccagaacagaggccggaagagaagaagccccagggctcccaagagacagagatgcatgcccgaagaggacctgcagagccagagcgaaacacagggactcgaaggtgctcaggctcctctggccgtggaagaagatgccagcagctctaccagcacctccagcagcttccctagcagctttccattcagctcctctagctctagcagcagctgttaccctctgatccccagcacacccgagaaggtgttcgccgacgacgagacacctaatccactgcagtctgcccagatcgcctgcagcagtacactggtggttgctagcctgcctctggaccagtctgatgagggaagcagcagccagaaagaggaaagccctagcacactccaggtgctgcccgatagcgagagcctgcctagaagcgagatctacaagaaaatgaccgacctggtgcagttcctcctgttcaagtaccagatgaaggaacccatcaccaaggccgaaatcctggaaagcgtgatcagaaactacgaggaccactttccactgctgttcagcgaggccagcgagtgcatgctgctcgtgtttagcatcgacgtgaagaaggtggaccccaccggccacagctttgtgctggttacaagcctgggactgacctacgacggcatgctgtccgatgtgcagagcatgcctaagaccggcatcctgatcctgattctgagcatcgtgttcatcgagggctactgcacccctgaggaagtgatttgggaagccctgaacatgatgggcctgtacgatggcatggaacacctgatctacggcgagcccagaaaactgctgacccaggactgggtgcaagagaactacctggaataccggcagatgcccggcagcgatcctgccagatatgagtttctgtggggccctagagcacatgccgagatccggaagatgagcctgctgaagttcctggccaaagtgaacggcagcgacccaatcagcttcccactttggtacgaagaggccctgaaggacgaggaagagagagcccaggatagaatcgccaccaccgacgacacaacagccatggcctctgcctcttctagcgccaccggcagctttagctaccccgagtgataa (SEQ ID NO: 45) modTBXT-modMAGEC2MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTEHELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVVADNHRWKYVNGEWVPGGKPQLQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITTLKIKYNPFAKAFLDAKERSDHKEMIKEPGDSQQPGYSQWGWLLPGTSTLCPPANPHSQFGGALSLSSTHSYDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLRMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTLGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGFPMYKGAAAATDIVDSQYDAAAQGHLIASWTPVSPPSMRGRKRRSPPVPGVPFRNVDNDSLTSVELEDWVDAQHPTDEEEEEASSASSTLYLVFSPSSFSTSSSLILGGPEEEEVPSGVIPNLTESIPSSPPQGPPQGPSQSPLSSCCSSFLWSSFSEESSSQKGEDTGTCQGLPDSESSFTYTLDEKVAKLVEFLLLKYEAEEPVTEAEMLMIVIKYKDYFPVILKRAREFMELLFGLALIEVGPDHFCVFANTVGLTDEGSDDEGMPENSLLIIILSVIFIKGNCASEEVIWEVLNAVGVYAGREHFVYGKPRELLTNVWVQGHYLEYWEVPHSSPLYYEFLWGPRAHSESIKKKVLEFLAKLNNTVPSFFPSWYKDALKDVEERVQATIDTADDATVMASESLSVMSSNVSFSE (SEQ ID NO: 46) modTBXT-modWT1atgtctagccctggaacagagtctgccggcaagagcctgcagtacagagtggaccatctgctgagcgccgtggaaaatgaactgcaggccggaagcgagaagggcgatcctacagagcacgagctgagagtcggcctggaagagtctgagctgtggctgcggttcaaagaactgaccaacgagatgatcgtgaccaagaacggcagacggatgttccccgtgctgaaagtgaacgtgtccggactggaccccaacgccatgtacagctttctgctggacttcgtggtggccgacaaccacagatggaaatacgtgaacggcgagtgggtgccaggcggaaaacctcaactgcaagcccctagctgcgtgtacattcaccctgacagccccaatttcggcgcccactggatgaaggcccctgtgtccttcagcaaagtgaagctgaccaacaagctgaacggcggaggccagatcatgctgaacagcctgcacaaatacgagcccagaatccacatcgtcagagtcggcggaccccagagaatgatcaccagccactgcttccccgagacacagtttatcgccgtgaccgcctaccagaacgaggaaatcaccacactgaagatcaagtacaaccccttcgccaaggccttcctggacgccaaagagcggagcgaccacaaagagatgatcaaagagcccggcgacagccagcagccaggctattctcaatggggatggctgctgccaggcaccagcacattgtgccctccagccaatcctcacagccagtttggaggcgccctgagcctgtctagcacccacagctacgacagataccccacactgcggagccacagaagcagcccctatccttctccttacgctcaccggaacaacagccccacctacagcgataatagccccgcctgtctgagcatgctgcagtcccacgataactggtccagcctgagaatgcctgctcacccttccatgctgcccgtgtctcacaatgcctctccacctaccagcagctctcagtaccctagcctttggagcgtgtccaatggcgccgtgacactgggatctcaggcagccgctgtgtctaatggactgggagcccagttcttcagaggcagccctgctcactacacccctctgacacatcctgtgtctgcccctagcagcagcggcttccctatgtataagggcgctgccgccgctaccgacatcgtggattctcagtatgatgccgccgcacagggacacctgatcgcctcttggacacctgtgtctccaccttccatgagaggcagaaagcggagaagcgacttcctgctgctgcagaaccctgcctctacctgtgtgcctgaaccagcctctcagcacaccctgagatctggccctggatgtctccagcagcctgaacagcagggcgttagagatcctggcggaatctgggccaaactgggagctgccgaagcctctgccgaatgtctgcagggcagaagaagcagaggcgccagcggatctgaacctcaccagatgggaagcgacgtgcacgacctgaatgctctgttgcctgccgtgccatctcttggcggaggcggaggatgtgctttgcctgtttctggtgctgcccagtgggctcccgtgctggattttgctcctcctggcgcttctgcctatggctctcttggaggacctgctcctccaccagctccacctccaccgccgcctccaccacctcacagctttatcaagcaagagccctcctggggcggagccgagcctcacgaaaaacagtgtctgagcgccttcaccgtgcactttttcggccagtttaccggcaccgtgggcgcctgtagatacggcccttttggaccaccaccacctagccaggcttctagcggacaggccagaatgttccccaacgctccttacctgcctagctgcctggaaagccagcctaccatcagaaaccagggcttcagcaccgtgaccttcgacggcatgcctagctatggccacacaccatctcaccacgccgctcagttccccaatcacagcttcaagcacgaggaccctatgggccagcagggatctctgggagagcagcagtatagcgtgccacctcctgtgtacggctgtcacacccctaccgatagctgcacaggcaatcaggctctgctgctgaggatgcctttcagcagcgacaacctgtaccagatgacaagccagctggaatgcatgatttggaaccagatgaacctgggcgccactctgaaaggcgtggccgctggatctagcagctccgtgaaatggacagccggccagagcaatcactccaccggctacgagagcgacaatcacaccatgcctatcctgtgtggggcccagtaccggattcacacacacggcgtgttcaggggcattcaggatgtgcgaagagtgcctggcgtggcccctacacttgtgggatctgccagcgaaaccagcgagaagcaccccttcatgtgcgcctatccaggctgcaacaagcggtacttcaagctgagccacctgaagatgcacagccggaagcacacaggcgagaagctgtaccagtgcgacttcaaggactgcgagcggagattcagctgcagcgaccagctgaagagacaccagagaaggcacaccggcgtgaagccctttcagtgcaagacctgccagcggaccttctcctggtccaaccacctgaaaacccacacaagaacccacaccggcaagaccatcgagaagcccttcagctgtagatggcccagctgccagaagaagttcgcccggtctaacgagctggtgcatcaccacaacatgcaccagaggaacatgaccaaactgcagctggtgctgtgatga (SEQ ID NO: 47)modTBXT_WT1MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTEHELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVVADNHRWKYVNGEWVPGGKPQLQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITTLKIKYNPFAKAFLDAKERSDHKEMIKEPGDSQQPGYSQWGWLLPGTSTLCPPANPHSQFGGALSLSSTHSYDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLRMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTLGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGFPMYKGAAAATDIVDSQYDAAAQGHLIASWTPVSPPSMRGRKRRSDFLLLQNPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAECLQGRRSRGASGSEPHQMGSDVHDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEKQCLSAFTVHFFGQFTGTVGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPTIRNQGFSTVTFDGMPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGNQALLLRMPFSSDNLYQMTSQLECMIWNQMNLGATLKGVAAGSSSSVKWTAGQSNHSTGYESDNHTMPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVGSASETSEKHPFMCAYPGCNKRYFKLSHLKMHSRKHTGEKLYQCDFKDCERRFSCSDQLKRHQRRHTGVKPFQCKTCQRTFSWSNHLKTHTRTHTGKTIEKPFSCRWPSCQKKFARSNELVHHHNMHQRNMTKLQLVL (SEQ ID NO: 48) modTBXT_WT1_(KRAS Mutations)agagtctgagctgtggctgcggttcaaagaactgaccaacgagatgatcgtgaccaagaacggcagacggatgttccccgtgctgaaagtgaacgtgtccggactggaccccaacgccatgtacagctttctgctggacttcgtggtggccgacaaccacagatggaaatacgtgaacggcgagtgggtgccaggcggaaaacctcaactgcaagcccctagctgcgtgtacattcaccctgacagccccaatttcggcgcccactggatgaaggcccctgtgtccttcagcaaagtgaagctgaccaacaagctgaacggcggaggccagatcatgctgaacagcctgcacaaatacgagcccagaatccacatcgtcagagtcggcggaccccagagaatgatcaccagccactgcttccccgagacacagtttatcgccgtgaccgcctaccagaacgaggaaatcaccacactgaagatcaagtacaaccccttcgccaaggccttcctggacgccaaagagcggagcgaccacaaagagatgatcaaagagcccggcgacagccagcagccaggctattctcaatggggatggctgctgccaggcaccagcacattgtgccctccagccaatcctcacagccagtttggaggcgccctgagcctgtctagcacccacagctacgacagataccccacactgcggagccacagaagcagcccctatccttctccttacgctcaccggaacaacagccccacctacagcgataatagccccgcctgtctgagcatgctgcagtcccacgataactggtccagcctgagaatgcctgctcacccttccatgctgcccgtgtctcacaatgcctctccacctaccagcagctctcagtaccctagcctttggagcgtgtccaatggcgccgtgacactgggatctcaggcagccgctgtgtctaatggactgggagcccagttcttcagaggcagccctgctcactacacccctctgacacatcctgtgtctgcccctagcagcagcggcttccctatgtataagggcgctgccgccgctaccgacatcgtggattctcagtatgatgccgccgcacagggacacctgatcgcctcttggacacctgtgtctccaccttccatgagaggcagaaagcggagaagcgacttcctgctgctgcagaaccctgcctctacctgtgtgcctgaaccagcctctcagcacaccctgagatctggccctggatgtctccagcagcctgaacagcagggcgttagagatcctggcggaatctgggccaaactgggagctgccgaagcctctgccgaatgtctgcagggcagaagaagcagaggcgccagcggatctgaacctcaccagatgggaagcgacgtgcacgacctgaatgctctgttgcctgccgtgccatctcttggcggaggcggaggatgtgctttgcctgtttctggtgctgcccagtgggctcccgtgctggattttgctcctcctggcgcttctgcctatggctctcttggaggacctgctcctccaccagctccacctccaccgccgcctccaccacctcacagctttatcaagcaagagccctcctggggcggagccgagcctcacgaaaaacagtgtctgagcgccttcaccgtgcactttttcggccagtttaccggcaccgtgggcgcctgtagatacggcccttttggaccaccaccacctagccaggcttctagcggacaggccagaatgttccccaacgctccttacctgcctagctgcctggaaagccagcctaccatcagaaaccagggcttcagcaccgtgaccttcgacggcatgcctagctatggccacacaccatctcaccacgccgctcagttccccaatcacagcttcaagcacgaggaccctatgggccagcagggatctctgggagagcagcagtatagcgtgccacctcctgtgtacggctgtcacacccctaccgatagctgcacaggcaatcaggctctgctgctgaggatgcctttcagcagcgacaacctgtaccagatgacaagccagctggaatgcatgatttggaaccagatgaacctgggcgccactctgaaaggcgtggccgctggatctagcagctccgtgaaatggacagccggccagagcaatcactccaccggctacgagagcgacaatcacaccatgcctatcctgtgtggggcccagtaccggattcacacacacggcgtgttcaggggcattcaggatgtgcgaagagtgcctggcgtggcccctacacttgtgggatctgccagcgaaaccagcgagaagcaccccttcatgtgcgcctatccaggctgcaacaagcggtacttcaagctgagccacctgaagatgcacagccggaagcacacaggcgagaagctgtaccagtgcgacttcaaggactgcgagcggagattcagctgcagcgaccagctgaagagacaccagagaaggcacaccggcgtgaagccctttcagtgcaagacctgccagcggaccttctcctggtccaaccacctgaaaacccacacaagaacccacaccggcaagaccatcgagaagcccttcagctgtagatggcccagctgccagaagaagttcgcccggtctaacgagctggtgcatcaccacaacatgcaccagaggaacatgaccaaactgcagctggtgctgaggggaagaaagaggcggtccaccgagtacaagctggtggttgttggagccgatggcgtgggaaagagcgccctgacaattcagctgatccagaaccacttcgtgcgcggcagaaagagaagatctacagagtataagctcgtggtcgtgggcgctgtcggagtgggaaaatctgccctgaccatccaactcattcagaatcactttgtgtgatga (SEQ ID NO: 49)modTBXT_WT1_(KRAS Mutations)MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTEHELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVVADNHRWKYVNGEWVPGGKPQLQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITTLKIKYNPFAKAFLDAKERSDHKEMIKEPGDSQQPGYSQWGWLLPGTSTLCPPANPHSQFGGALSLSSTHSYDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLRMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTLGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGFPMYKGAAAATDIVDSQYDAAAQGHLIASWTPVSPPSMRGRKRRSDFLLLQNPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAECLQGRRSRGASGSEPHQMGSDVHDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEKQCLSAFTVHFFGQFTGTVGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPTIRNQGFSTVTFDGMPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGNQALLLRMPFSSDNLYQMTSQLECMIWNQMNLGATLKGVAAGSSSSVKWTAGQSNHSTGYESDNHTMPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVGSASETSEKHPFMCAYPGCNKRYFKLSHLKMHSRKHTGEKLYQCDFKDCERRFSCSDQLKRHQRRHTGVKPFQCKTCQRTFSWSNHLKTHTRTHTGKTIEKPFSCRWPSCQKKFARSNELVHHHNMHQRNMTKLQLVLRGRKRRSTEYKLVVVGADGVGKSALTIQLIQNHFVRGRKRRSTEYKLVVVGAVGVGKSALTIQLIQNHFV (SEQ ID NO: 50) modWT1-modFBPatggactttctgctgctgcagaaccctgccagcacctgtgttccagaacctgcctctcagcacaccctgagatctggccctggatgtctccagcagcctgaacagcagggcgttagagatcctggcggaatctgggccaaactgggagccgctgaagcctctgccgaatgtctgcagggcagaagaagcagaggcgccagcggatctgaacctcaccagatgggaagcgacgtgcacgacctgaatgctctgctgcctgccgtgccatctcttggcggaggcggaggatgtgctttgcctgtttctggtgctgcccagtgggctcccgtgctggattttgctcctcctggcgcttctgcctatggctctcttggaggacctgctcctccaccagctccacctccaccgccgcctccaccacctcacagctttatcaagcaagagccctcctggggcggagccgagcctcacgaaaaacagtgtctgagcgccttcaccgtgcactttttcggccagtttaccggcacagtgggcgcctgtagatacggcccttttggaccaccaccacctagccaggctagctctggacaggccagaatgttccccaacgctccctacctgcctagctgcctggaaagccagcctaccatcagaaaccagggcttcagcaccgtgaccttcgacggcatgcctagctatggccacacaccatctcaccacgccgctcagttccccaatcacagcttcaagcacgaggaccctatgggccagcagggatctctgggagagcagcagtatagcgtgccacctcctgtgtacggctgtcacacccctaccgatagctgcacaggcaatcaggccctgctgctgaggatgcccttcagcagcgacaacctgtaccagatgacaagccagctggaatgcatgatctggaaccagatgaacctgggcgccacactgaaaggcgtggccgctggatctagcagcagcgtgaaatggacagccggccagagcaatcactccaccggctacgagtccgacaaccacaccatgcctattctgtgcggagcccagtacagaatccacacacacggcgtgttccggggcattcaggatgtgcgaagagtgcctggcgtggcccctacacttgtgggatctgcctctgagacaagcgagaagcaccccttcatgtgcgcctatcctggctgcaacaagcggtacttcaagctgagccacctgaagatgcacagccggaagcacacaggcgagaagctgtaccagtgcgacttcaaggactgcgagcggagattcagctgcagcgaccagctgaagagacaccagagaaggcacaccggcgtgaagcccttccagtgcaagacctgccagcggacctttagctggtccaaccacctgaaaacccacacaagaacccacaccggcaagaccatcgagaagcctttcagctgtagatggcccagctgccagaagaagttcgcccggtctaacgagctggtgcatcaccacaacatgcaccagaggaacatgaccaaactgcagctggtgctgaggggaagaaagcggagaagcgcccagagaatgaccacacagttgctgctgctcctcgtgtgggttgccgttgtgggagaagtgcagaccagaatcgcctgggccagaaccgagctgctgaacgtgtgcatgaacgccaagcaccacaagaagaagcccgatcctgaggacaagctgcacgagcagtgtcggccttggagaaagaacgcctgctgtagcaccaacaccagccaagaggcccacaagaacgtgtcctacctgtaccggttcaactggaaccactgcggcgagatgacacccgcctgcaagagacacttcatccaggatacctgcctgtacgagtgcagccccaatctcggcccctggattcagcaagtggaccagagctggcggaaagaactggtcctgaatgtgcccctgtgcaaagaggattgcgagcagtggtgggaagattgcagaaccagctacacatgcaagagcaactggcacaaaggctggaactggaccagcggcttcaacaagtgtgccgtgggagctgcctgtcagcctttccacttctactttcacacacccaccgtgctgtgcaacaagatctggacccacagctacaaggtgtccaactacagcagaggcagcggccggtgtatccagatgtggttcgatcccgccaagggcaaccccaatgaggaagtggccagattctacgccgctgccatgtctggtgcaggaccttgggctgcttggccctttctgctttcactggccctgatgctgctgtggctgctgagctgataa (SEQ ID NO: 51)modWT1-modFBPMDFLLLQNPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAECLQGRRSRGASGSEPHQMGSDVHDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEKQCLSAFTVHFFGQFTGTVGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPTIRNQGFSTVTFDGMPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGNQALLLRMPFSSDNLYQMTSQLECMIWNQMNLGATLKGVAAGSSSSVKWTAGQSNHSTGYESDNHTMPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVGSASETSEKHPFMCAYPGCNKRYFKLSHLKMHSRKHTGEKLYQCDFKDCERRFSCSDQLKRHQRRHTGVKPFQCKTCQRTFSWSNHLKTHTRTHTGKTIEKPFSCRWPSCQKKFARSNELVHHHNMHQRNMTKLQLVLRGRKRRSAQRMTTQLLLLLVWVAVVGEVQTRIAWARTELLNVCMNAKHHKKKPDPEDKLHEQCRPWRKNACCSTNTSQEAHKNVSYLYRFNWNHCGEMTPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKELVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFHTPTVLCNKIWTHSYKVSNYSRGSGRCIQMWFDPAKGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS (SEQ ID NO: 52) modPSMA-modTDGF1atgtggaatctgctgcacgagacagatagcgccgtggctaccgttagaaggcccagatggctttgtgctggcgctctggttctggctggcggcttttttctgctgggcttcctgttcggctggttcatcaagagcagcaacgaggccaccaacatcacccctaagcacaacatgaaggcctttctggacgagctgaaggccgagaatatcaagaagttcctgtacaacttcacgcacatccctcacctggccggcaccgagcagaattttcagctggccaagcagatccagagccagtggaaagagttcggcctggactctgtggaactggcccactacgatgtgctgctgagctaccccaacaagacacaccccaactacatcagcatcatcaacgaggacggcaacgagatcttcaacaccagcctgttcgagcctccacctcctggctacgagaacgtgtccgatatcgtgcctccattcagcgctttcagcccacagcggatgcctgagggctacctggtgtacgtgaactacgccagaaccgaggacttcttcaagctggaatgggacatgaagatcagctgcagcggcaagatcgtgatcgcccggtacagaaaggtgttccgcgagaacaaagtgaagaacgcccagctggcaggcgccaaaggcgtgatcctgtatagcgaccccgccgactattttgcccctggcgtgaagtcttaccccgacggctggaattttcctggcggcggagtgcagcggcggaacatccttaatcttaacggcgctggcgaccctctgacacctggctatcctgccaatgagtacgcctacagacacggaattgccgaggctgtgggcctgccttctattcctgtgcaccctgtgcggtactacgacgcccagaaactgctggaaaagatgggcggaagcgcccctcctgactcttcttggagaggctctctgaaggtgccctacaatgtcggcccaggcttcaccggcaacttcagcacccagaaagtgaaaatgcacatccacagcaccaacgaagtgacccggatctacaacgtgatcggcacactgagaggcgccgtggaacccgacaaatacgtgatcctcggcggccacagagacagctgggtgttcggaggaatcgaccctcaatctggcgccgctgtggtgtatgagatcgtgcggtctttcggcaccctgaagaaagaaggatggcggcccagacggaccatcctgtttgcctcttgggacgccgaggaatttggcctgctgggatctacagagtgggccgaagagaacagcagactgctgcaagaaagaggcgtggcctacatcaacgccgacagcagcatcgagggcaactacaccctgcggatcgattgcacccctctgatgtacagcctggtgcacaacctgaccaaagagctgaagtcccctgacgagggctttgagggcaagagcctgtacaagagctggaccaagaagtccccatctcctgagttcagcggcatgcccagaatctctaagctggaaagcggcaacaacttcgaggtgttcttccagcggctgggaatcgcctctggaatcgccagatacaccaagaactgggagacaaacaagttctccggctatcccctgtaccacagcgtgtacgagacatacgagctggtggaaaagttctacgaccccatgttcaagtaccacctgacagtggcccaagtgcgcggaggcatggtgttcgaactggccaatagcatcgtgctgcccttcaactgcagagactacgccgtggtgctgcggaagtacgccgacaagatctacagcatcagcatgaagcacccgcaagagatgaagacctacagcgtgtccttcgactccctgttcttcgccgtgaagaacttcaccaagatcgccagcaagttcagcgagcggctgcaggacttcgacaagagcaaccctatcgtgctgaggatgatgaacgaccagctgatgttcctggaacgggccttcatcaaccctctgggactgcccgacagacccttctacaggcacgtgatctgtgcccctagcagccacaacaaatacgccggcgagagcttccccggcatctacgatgccctgttcgacatcgagagcaacgtgaaccctagcaaggcctggggcgaagtgaagagacagatctacgtggccgcattcacagtgcaggccgctgccgaaacactgtctgaagtggccagaggccggaagagaagatccgactgcagaaagatggcccggttcagctactccgtgatctggatcatggccatctccaaggccttcgagctgagactggttgccggactgggccaccaagagtttgccagacctagctggggctatctggccttccgggacgatagcatctggccccaagaggaacctgccatcagacccagatctagccagcgggtgccacctatggaaatccagcacagcaaagaactgaaccggacctgctgcctgaacggcagaacctgtatgctgggcagcttctgcgcctgtcctcctagcttctacggccggaattgcgagcacgacgtgcggaaagaaaactgcggcagcgtgccacacgatacctggctgcctaagaaatgcagcctgtgcaagtgttggcacggccagctgcggtgtttccccagagcttttctgcccgtgtgtgacggcctggtcatggatgaacacctggtggccagcagaacccctgagcttcctccaagcgccaggaccaccacctttatgctcgtgggcatctgcctgagcatccagagctactactgatga (SEQ ID NO: 53)modPSMA-modTDGF1MWNLLHETDSAVATVRRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTHIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQRMPEGYLVYVNYARTEDFFKLEWDMKISCSGKIVIARYRKVFRENKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNFPGGGVQRRNILNLNGAGDPLTPGYPANEYAYRHGIAEAVGLPSIPVHPVRYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDKYVILGGHRDSWVFGGIDPQSGAAVVYEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRIDCTPLMYSLVHNLTKELKSPDEGFEGKSLYKSWTKKSPSPEFSGMPRISKLESGNNFEVFFQRLGIASGIARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFNCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFFAVKNFTKIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFINPLGLPDRPFYRHVICAPSSHNKYAGESFPGIYDALFDIESNVNPSKAWGEVKRQIYVAAFTVQAAAETLSEVARGRKRRSDCRKMARFSYSVIWIMAISKAFELRLVAGLGHQEFARPSWGYLAFRDDSIWPQEEPAIRPRSSQRVPPMEIQHSKELNRTCCLNGRTCMLGSFCACPPSFYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPRAFLPVCDGLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYY (SEQ ID NO: 54) modWT1-modClaudin 18atggactttctgctgctgcagaaccctgccagcacctgtgttccagaacctgcctctcagcacaccctgagatctggccctggatgtctccagcagcctgaacagcagggcgttagagatcctggcggaatctgggccaaactgggagccgctgaagcctctgccgaatgtctgcagggcagaagaagcagaggcgccagcggatctgaacctcaccagatgggaagcgacgtgcacgacctgaatgctctgctgcctgccgtgccatctcttggcggaggcggaggatgtgctttgcctgtttctggtgctgcccagtgggctcccgtgctggattttgctcctcctggcgcttctgcctatggctctcttggaggacctgctcctccaccagctccacctccaccgccgcctccaccacctcacagctttatcaagcaagagccctcctggggcggagccgagcctcacgaaaaacagtgtctgagcgccttcaccgtgcactttttcggccagtttaccggcacagtgggcgcctgtagatacggcccttttggaccaccaccacctagccaggctagctctggacaggccagaatgttccccaacgctccctacctgcctagctgcctggaaagccagcctaccatcagaaaccagggcttcagcaccgtgaccttcgacggcatgcctagctatggccacacaccatctcaccacgccgctcagttccccaatcacagcttcaagcacgaggaccctatgggccagcagggatctctgggagagcagcagtatagcgtgccacctcctgtgtacggctgtcacacccctaccgatagctgcacaggcaatcaggccctgctgctgaggatgcccttcagcagcgacaacctgtaccagatgacaagccagctggaatgcatgatctggaaccagatgaacctgggcgccacactgaaaggcgtggccgctggatctagcagcagcgtgaaatggacagccggccagagcaatcactccaccggctacgagtccgacaaccacaccatgcctattctgtgcggagcccagtacagaatccacacacacggcgtgttccggggcattcaggatgtgcgaagagtgcctggcgtggcccctacacttgtgggatctgcctctgagacaagcgagaagcaccccttcatgtgcgcctatcctggctgcaacaagcggtacttcaagctgagccacctgaagatgcacagccggaagcacacaggcgagaagctgtaccagtgcgacttcaaggactgcgagcggagattcagctgcagcgaccagctgaagagacaccagagaaggcacaccggcgtgaagcccttccagtgcaagacctgccagcggacctttagctggtccaaccacctgaaaacccacacaagaacccacaccggcaagaccatcgagaagcctttcagctgtagatggcccagctgccagaagaagttcgcccggtctaacgagctggtgcatcaccacaacatgcaccagaggaacatgaccaaactgcagctggtgctgaggggaagaaagcggagatctgccgtgacagcctgtcagagcctgggctttgtggtgtccctgatcgagatcgtgggcatcattgccgctacctgcatggaccagtggtctacccaggacctgtacaacaaccctgtgaccgccgtgttcaactaccaaggcctgtggcacagctgcatgagagagagcagcggcttcaccgagtgcagaggctacttcaccctgctggaactgcctgccatgctgcaggctgtgcaggcccttatgatcgtgggaattgtgctgggagccatcggcctgctggtgtccattttcgccctgaagtgcatccggatcggcagcatggaagatagcgccaaggccaacatgaccctgaccagcggcatcatgttcatcgtgtccggcctgtgcgccattgctggcgtgtccgtgtttgccaatatgctcgtgaccaacttctggctgagcaccgccaacatgtacaccggcatgggcgagatggtgcagaccgtgcagacacggtacacatttggcgccgctctgtttgtcggatgggttgcaggcggactgacactgattggcggcgtgatgatgtgtatcgcctgcagaggactggcccctgaggaaacaaactacaaggccgtgtactaccacgcctccggacacagcgtggcatacaaacctggcggctttaaggccagcaccggcttcggcagcaacaccaagaacaagaagatctacgacggcggagcacacaccgaggatgaggtgcagagctaccccagcaagcacgactacgtgtgatga (SEQ ID NO: 55)modWT1-modClaudin 18MDFLLLQNPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAECLQGRRSRGASGSEPHQMGSDVHDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEKQCLSAFTVHFFGQFTGTVGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPTIRNQGFSTVTFDGMPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGNQALLLRMPFSSDNLYQMTSQLECMIWNQMNLGATLKGVAAGSSSSVKWTAGQSNHSTGYESDNHTMPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVGSASETSEKHPFMCAYPGCNKRYFKLSHLKMHSRKHTGEKLYQCDFKDCERRFSCSDQLKRHQRRHTGVKPFQCKTCQRTFSWSNHLKTHTRTHTGKTIEKPFSCRWPSCQKKFARSNELVHHHNMHQRNMTKLQLVLRGRKRRSAVTACQSLGRNSLIEIVGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWHSCMRESSGFTECRGYFTLLELPAMLQAVQALMIVGIVLGAIGLLVSPEETNYKAVYYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGAHTEDEVQSYPSKHDYV (SEQ ID NO: 56)modPSMA-modLY6Katgtggaatctgctgcacgagacagatagcgccgtggctaccgttagaaggcccagatggctttgtgctggcgctctggttctggctggcggcttttttctgctgggcttcctgttcggctggttcatcaagagcagcaacgaggccaccaacatcacccctaagcacaacatgaaggcctttctggacgagctgaaggccgagaatatcaagaagttcctgtacaacttcacgcacatccctcacctggccggcaccgagcagaattttcagctggccaagcagatccagagccagtggaaagagttcggcctggactctgtggaactggcccactacgatgtgctgctgagctaccccaacaagacacaccccaactacatcagcatcatcaacgaggacggcaacgagatcttcaacaccagcctgttcgagcctccacctcctggctacgagaacgtgtccgatatcgtgcctccattcagcgctttcagcccacagcggatgcctgagggctacctggtgtacgtgaactacgccagaaccgaggacttcttcaagctggaatgggacatgaagatcagctgcagcggcaagatcgtgatcgcccggtacagaaaggtgttccgcgagaacaaagtgaagaacgcccagctggcaggcgccaaaggcgtgatcctgtatagcgaccccgccgactattttgcccctggcgtgaagtcttaccccgacggctggaattttcctggcggcggagtgcagcggcggaacatccttaatcttaacggcgctggcgaccctctgacacctggctatcctgccaatgagtacgcctacagacacggaattgccgaggctgtgggcctgccttctattcctgtgcaccctgtgcggtactacgacgcccagaaactgctggaaaagatgggcggaagcgcccctcctgactcttcttggagaggctctctgaaggtgccctacaatgtcggcccaggcttcaccggcaacttcagcacccagaaagtgaaaatgcacatccacagcaccaacgaagtgacccggatctacaacgtgatcggcacactgagaggcgccgtggaacccgacaaatacgtgatcctcggcggccacagagacagctgggtgttcggaggaatcgaccctcaatctggcgccgctgtggtgtatgagatcgtgcggtctttcggcaccctgaagaaagaaggatggcggcccagacggaccatcctgtttgcctcttgggacgccgaggaatttggcctgctgggatctacagagtgggccgaagagaacagcagactgctgcaagaaagaggcgtggcctacatcaacgccgacagcagcatcgagggcaactacaccctgcggatcgattgcacccctctgatgtacagcctggtgcacaacctgaccaaagagctgaagtcccctgacgagggctttgagggcaagagcctgtacaagagctggaccaagaagtccccatctcctgagttcagcggcatgcccagaatctctaagctggaaagcggcaacaacttcgaggtgttcttccagcggctgggaatcgcctctggaatcgccagatacaccaagaactgggagacaaacaagttctccggctatcccctgtaccacagcgtgtacgagacatacgagctggtggaaaagttctacgaccccatgttcaagtaccacctgacagtggcccaagtgcgcggaggcatggtgttcgaactggccaatagcatcgtgctgcccttcaactgcagagactacgccgtggtgctgcggaagtacgccgacaagatctacagcatcagcatgaagcacccgcaagagatgaagacctacagcgtgtccttcgactccctgttcttcgccgtgaagaacttcaccaagatcgccagcaagttcagcgagcggctgcaggacttcgacaagagcaaccctatcgtgctgaggatgatgaacgaccagctgatgttcctggaacgggccttcatcaaccctctgggactgcccgacagacccttctacaggcacgtgatctgtgcccctagcagccacaacaaatacgccggcgagagcttccccggcatctacgatgccctgttcgacatcgagagcaacgtgaaccctagcaaggcctggggcgaagtgaagagacagatctacgtggccgcattcacagtgcaggccgctgccgaaacactgtctgaagtggccagaggccggaagagaagaagtgctctgctggcactgctgctggtggtggctttgcctagagtgtggaccgacgccaatctgacagtgcggcagagagatcctgaggacagccagagaaccgacgacggcgataacagagtgtggtgccacgtgtgcgagcgcgagaataccttcgagtgtcagaaccccagacggtgcaagtggaccgagccttactgtgtgatcgccgccgtgaaaatcttcccacggttcttcatggtggtcaagcagtgcagcgctggctgtgccgctatggaaagacccaagcctgaggaaaagcggttcctgctcgaggaacccatgctgttcttctacctgaagtgctgcaaaatctgctactgcaacctggaaggccctcctatcaacagcagcgtcctgaaagaatatgccggcagcatgggcgagtcttgtggtggactgtggctggccattctgctgctgcttgcctctattgccgcctctctgagcctgagctgatga(SEQ ID NO: 57) modPSMA-modLY6KMWNLLHETDSAVATVRRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTHIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQRMPEGYLVYVNYARTEDFFKLEWDMKISCSGKIVIARYRKVFRENKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNFPGGGVQRRNILNLNGAGDPLTPGYPANEYAYRHGIAEAVGLPSIPVHPVRYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDKYVILGGHRDSWVFGGIDPQSGAAVVYEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRIDCTPLMYSLVHNLTKELKSPDEGFEGKSLYKSWTKKSPSPEFSGMPRISKLESGNNFEVFFQRLGIASGIARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFNCRDYAWLRKYADKIYSISMKHPQEMKTYSVSFDSLFFAVKNFTKIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFINPLGLPDRPFYRHVICAPSSHNKYAGESFPGIYDALFDIESNVNPSKAWGEVKRQIYVAAFTVQAAAETLSEVARGRKRRSALLALLLWALPRVWTDANLTVRQRDPEDSQRTDDGDNRVWCHVCERENTFECQNPRRCKWTEPYCVIAAVKIFPRFFMWKQCSAGCAAMERPKPEEKRFLLEEPMLFFYLKCCKICYCNLEGPPINSSVLKEYAGSMGESCGGLWLAILLLLASIAASLSLS (SEQ ID NO: 58) modBORISatggccgctacagagattagcgtgctgagcgagcagttcaccaagatcaaagaactgaagctgatgctcgagaagggcctgaagaaagaagagaaggacggcgtctgccgcgagaagaaccacagaagcccatctgagctggaagcccagagaacctctggcgccttccaggacagcatcctggaagaggaagtggaactggttctggcccctctggaagagagcaagaagtacatcctgacactgcagaccgtgcacttcacctctgaagccgtgcagctccaggacatgagcctgctgtctatccagcagcaagagggcgtgcaggttgtggttcagcaacctggacctggactgctgtggctgcaagagggacctagacagagcctgcagcagtgtgtggccatcagcatccagcaagagctgtactcccctcaagagatggaagtgctgcagtttcacgccctggaagaaaacgtgatggtggccatcgaggacagcaagctggctgtgtctctggccgaaaccaccggcctgatcaagctggaagaagaacaagagaagaatcagctgctcgccgaaaagaccaaaaagcaactgttcttcgtggaaaccatgagcggcgacgagcggagcgacgaaatcgtgctgaccgtgtccaacagcaacgtcgaggaacaagaggaccagcctacagcctgtcaggccgatgccgagaaagccaagtttaccaagaaccagagaaagaccaagggcgccaagggcaccttccactgcaacgtgtgcatgttcaccagcagccggatgagcagcttcaactgccacatgaagacccacaccagcgagaagccccacctgtgccatctgtgcctgaaaaccttccggaccgtgactctgctgtggaactacgtgaacacccacacaggcacccggccttacaagtgcaacgactgcaacatggccttcgtgaccagcggagaactcgtgcggcacagaagatacaagcacacccacgagaaacccttcaagtgcagcatgtgcaaatacgccagcatggaagcctccaagctgaagtgtcacgtgcggagccatacaggcgagcaccctttccagtgctgccagtgtagctacg cctccagggacacctataagctgaagcggcacatgagaacccactctggggagaagccttacgagtgccacatctgccacaccagattcacccagagcggcaccatgaagattcacatcctgcagaaacacggcaagaacgtgcccaagtaccagtgtcctcactgcgccaccattatcgccagaaagtccgacctgcgggtgcacatgaggaatctgcacgcctattctgccgccgagctgaaatgcagatactgcagcgccgtgttccacaagagatacgccctgatccagcaccagaaaacccacaagaacgagaagcggtttaagtgcaagcactgctcctacgcctgcaagcaagagcgccacatgatcgcccacatccacacacacaccggcgaaaagcctttcacctgtctgagctgcaacaagtgcttccggcagaaacagctgctgaacgcccacttcagaaagtaccacgacgccaacttcatccccaccgtgtacaagtgctccaagtgcggcaagggcttcagccggtggatcaatctgcaccggcacctggaaaagtgcgagtctggcgaagccaagtctgccgcctctggcaagggcagaagaacccggaagagaaagcagaccattctgaaagaggccaccaagagccagaaagaagccgccaagcgctggaaagaggctgccaacggcgacgaagctgccgctgaagaagccagcacaacaaagggcgaacagttccccgaagagatgttccccgtggcctgcagagaaaccacagccagagtgaagcaagaggtggaccagggcgtcacatgcgagatgctgctgaataccatggacaagtgatga (SEQ ID NO: 59)modBORISMAATEISVLSEQFTKIKELKLMLEKGLKKEEKDGVCREKNHRSPSELEAQRTSGAFQDSILEEEVELVLAPLEESKKYILTLQTVHFTSEAVQLQDMSLLSIQQQEGVQWVQQPGPGLLWLQEGPRQSLQQCVAISIQQELYSPQEMEVLQFHALEENVMVAIEDSKLAVSLAETTGLIKLEEEQEKNQLLAEKTKKQLFFVETMSGDERSDEIVLTVSNSNVEEQEDQPTACQADAEKAKFTKNQRKTKGAKGTFHCNVCMFTSSRMSSFNCHMKTHTSEKPHLCHLCLKTFRTVTLLWNYVNTHTGTRPYKCNDCNMAFVTSGELVRHRRYKHTHEKPFKCSMCKYASMEASKLKCHVRSHTGEHPFQCCQCSYASRDTYKLKRHMRTHSGEKPYECHICHTRFTQSGTMKIHILQKHGKNVPKYQCPHCATIIARKSDLRVHMRNLHAYSAAELKCRYCSAVFHKRYALIQHQKTHKNEKRFKCKHCSYACKQERHMIAHIHTHTGEKPFTCLSCNKCFRQKQLLNAHFRKYHDANFIPTVYKCSKCGKGFSRWINLHRHLEKCESGEAKSAASGKGRRTRKRKQTILKEATKSQKEAAKRWKEAANGDEAAAEEASTTKGEQFPEEMFPVACRETTARVKQEVDQGVTCEMLLNTMDK (SEQ ID NO: 60) modMesothelinatggcattgcctacagctagacctctgctgggcagctgtggaacaccagctctgggaagcctgctgtttctgctgttcagcctcggatgggtgcagccttctagaacactggccggcgagacaggacaagaagctgctcctcttgacggcgtgctggccaatcctcctaatatcagctctctgagccccagacagctgctcggctttccttgtgccgaagtgtctggcctgagcaccgagagagtgtgggaacttgctgtggccctggctcagaaaaacgtgaagctgagcacagagcagctgagatgtctggcccaccagctgagtgaacctccagaggatctggatgccctgcctctggacctgctgctgttcctgaatcctgacgcctttagcggccctcaggcctgcaccagattcttcagcagaatcaccaaggccaatgtggatctgctgcccagaggcgcccctgagagacaaagacttctgcctgctgctctggcctgttggggcgttagaggatctctgctgtctgaggccgatgtgctggctcttggaggcctggcttgtaacctgcctggcagatttgtggccgagtctgctgaggtgctgctgcctagactggtgtcctgtcctggacctctggatcaggaccagcaagaagccgctagagctgcacttcaaggcggcggacctccttatggacctcctctgacttggagcgtgtccaccatggacgctctgagaggactgctgcctgttctgggccagcctatcatccggtctatccctcagggaattgtggccgcttggcggcagagaagcttcagagatccctcttggagacagcccaagcagaccatcctgtggcctcggttcagatgggaagtcgagaaaaccgcctgtcctagcggcaagaaggccagagagatcgacgagagcctgatcttctacaagaagtgggaactcgaggcctgcgtggacgctgctctgctggctacacagatggacagagtgaacgctatccccttcacctatgagcagctggacgtgctgaagcacaagctggatgagctgtaccctcagggctaccccgagtctgtgattcagcacctgggctacctgtttctgaagatgagccccgaggacatccggaagtggaacgtgaccagcctggaaaccctgaaggccctgctggaagtgaacaagggccacgagatgtccccacaggctcctagaaggcctctgcctcaagtggccacactgatcgacagattcgtgaaaggcaggggccagctggacaaggacaccctggatacactgaccgccttctatcccggctatctgtgcagcctgtctcctgaggaactgtcctctgtgcctcctagctctatttgggctgtgcggcctcaggacctggatacctgtgatcctagacagctggatgtcctgtatcctaaggctcggctggccttccagaacatgaacggcagcgagtacttcgtgaagatccagttcttccttggcggcgctcccaccgaggatctgaaagctctgtcccagcagaatgtgtctatggacctggccacctttatgaagctgcggaccgatgctgtgctgcctctgacagtggccgaggtgcaaaaactgctgggccctcatgtggaaggactgaaggccgaagaacggcacagacccgtcagagactggattctgagacagcggcaggacgacctggacacactggaacttggactgcaaggcggcatccccaatggctacctggtgctggatctgagcgtgcaagaggccctctctggcacaccttgtttgctcggacctggaccagtgctgacagtgttggctctgctgctggcctctacactggcctgataa (SEQ ID NO: 61)modMesothelinMALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVWELAVALAQKNVKLSTEQLRCLAHQLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVLALGGLACNLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPLTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSFRDPSWRQPKQTILWPRFRWEVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQFFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLELGLQGGIPNGYLVLDLSVQEALSGTPCLLGPGPVLTVLALLLASTLA (SEQ ID NO: 62)modFAP-modClaudin 18cctgaacgtgaccttcagctacaagatattcttccccaactggatctccggccaagagtacctgcaccagagcgccgacaacaacatcgtgctgtacaacatcgagacaggccagagctacaccatcatgagcaaccggaccatgaagtccgtgaacgccagcaactacggactgagccccgattggcagttcgtgtacctggaaagcgactacagcaagctgtggcggtacagctacaccgccacctactacatctacgacctgagcaacggcgagttcgtgaagggcaacgagctgccccatcctatccagtacctgtgttggagccctgtgggctccaagctggcctacgtgtaccagaacaacatctacctgaagcagcggcctggcgaccctccattccagatcaccttcaacggcagagagaacaagatctttaacggcatccccgactgggtgtacgaggaagagatgctggccaccaaatacgccctgtggtggtcccctaacggcaagtttctggcctatgccgacttcaacgacacagacatccccgtgatcgcctacagctactacggcaatgagcagtaccccaggaccatcaacatcagctaccccaaagccggcgctaagaaccctgtcgtgcggatcttcatcatcgacaccacctatcctgtgtacgtgggccctcaagaggtgccagtgcctgccatgattgccagcagcgactactacttcagctggctgacctgggtcaccgacgagcgagtttgtctgcagtggctgaagcgggtgcagaacatcagcgtgctgagcatctgcgacttcagaaaggactggcagacatgggactgccccaacacacagcagcacatcgaggaaagcagaaccggctgggctggcggcttctttgtgtctacccctgtgttcagctacgacgccatcctgtactataagatcttcagcgacaaggacggctacaagcacatccactacatcaagtacaccgtcgagaacgtgatccagattaccagcggcaagtgggaagccatcaatatcttcagagtgatccagtacagcctgttctacagcagcaacgagttcgaggaataccccggcagacggaacatctacagaatcagcatcggcagctacccgcctagcaagaaatgcgtgacctgccacctgagaaaagagcggtgccagtactacacagccagcttctccaactacgccaagtactacgccctcgtgtgttacggccctggcatccctatcagcacactgcacgatggcagaaccgaccaagagatcaagatcctggaagaaaacaaagagctggaaaacgccctgaagaacatccagctgcctaaagaggaaatcaagaagctggaagtcgacgagatcaccctgtggtacaagatgatcctgcctcctcagttcgaccggtccaagaagtaccctctgctgatccaggtgtacggcggaccttgttctcagtctgtgcgctccgtgttcgccgtgaattggatcagctatctggccagcaaagaaggcatggttatcgccctggtggacggcagaggcacagcttttcaaggcgacaagctgctgtacgccgtgtatcagaaactgggcgtgtacgaagtggaagatcagatcaccgccgtgcggaagttcatcgagatgggcttcatcgacgagaagcggatcgccatctggggctggtcttacggcggctatattagctctctggccctggcctctggcaccggcctgtttaagtgtggaattgccgtggctcccgtgtccagctgggagtactataccagcgtgtacaccgagcggttcatgggcctgcctaccaaggacgacaacctggaacactacaagaactctaccgtgatggccagagccgagtacttccggaacgtggactacctgctgattcacggcaccgccgacgacaacgtgcacttccaaaacagcgcccagatcgctaaggccctcgtgaatgcccaggtggactttcaggccatgtggtacagcgaccagaaccacggactgtctggcctgagcaccaaccacctgtacacccacatgacccactttctgaaacagtgcttcagcctgagcgaccggggcagaaagagaagatctgccgtcacagcctgtcagagcctgggctttgtggtgtccctgatcgagatcgtgggcatcattgccgctacctgcatggaccagtggtctacccaggacctgtataacaaccccgtgaccgccgtgttcaactaccaaggcctgtggcacagctgcatgagagagagcagcggcttcaccgagtgcaggggctactttaccctgctggaactgccagccatgctgcaggctgtgcaggcccttatgatcgtgggaattgtgctgggcgccatcggcctgctggtgtctatttttgccctgaagtgcatccggatcggcagcatggaagatagcgccaaggccaacatgaccctgacctccggcatcatgttcatcgtgtccggcctgtgtgccattgcaggcgtgtccgtgtttgccaatatgctcgtgaccaacttctggctgtccaccgccaacatgtacaccggcatgggcgagatggtgcagaccgtgcagacacggtacacatttggcgccgctctgtttgtcggatgggttgcaggcggactgactctgattggcggcgtgatgatgtgtatcgcctgcagaggactggcccctgaggaaacaaactacaaggccgtgtactaccacgccagcggacacagcgtggcatacaaaccaggcggctttaaggccagcacaggcttcggcagcaacaccaagaacaagaagatctacgacggcggagcccataccgaggatgaggtgcagagctaccctagcaagcacgactacgtgtgatga (SEQ ID NO: 63)modFAP-modClaudin 18MKTLVKIVFGVATSAVLALLVMCIVLHPSRVHNSEENTMRALTLKDILNVTFSYKIFFPNWISGQEYLHQSADNNIVLYNIETGQSYTIMSNRTMKSVNASNYGLSPDWQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVKGNELPHPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYADFNDTDIPVIAYSYYGNEQYPRTINISYPKAGAKNPVVRIFIIDTTYPVYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNISVLSICDFRKDWQTWDCPNTQQHIEESRTGWAGGFFVSTPVFSYDAILYYKIFSDKDGYKHIHYIKYTVENVIQITSGKWEAINIFRVIQYSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSNYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYQKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYISSLALASGTGLFKCGIAVAPVSSWEYYTSVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMINYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDRGRKRRSAVTACQSLGFWSLIEIVGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWHSCMRESSGFTECRGYFTLLELPAMLQAVQALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWLSTANMYTGMGEMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVYYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGAHTEDEVQSYPSKHDYV (SEQ ID NO: 64) modPRAME-modTBXTatggaaagaagaaggctctggggcagcatccagagccggtacatcagcatgagcgtgtggacaagccctcggagactggtggaactggctggacagagcctgctgaaggatgaggccctggccattgctgctctggaactgctgcctagagagctgttccctcctctgttcatggccgccttcgacggcagacacagccagacactgaaagccatggtgcaggcctggcctttcacctgtctgcctctgggagtgctgatgaagggccagcatctgcacctggaaaccttcaaggccgtgctggatggcctggatgtgctgctggctcaagaagtgcggcctcggcgttggaaactgcaggttctggatctgctgaagaacagccaccaggatttctggaccgtttggagcggcaacagagccagcctgtacagctttcctgagcctgaagccgctcagcccatgaccaagaaaagaaaggtggacggcctgagcaccgaggccgagcagccttttattcccgtggaagtgctggtggacctgttcctgaaagaaggcgcctgcgacgagctgttcagctacctgaccgagaaagtgaagcagaagaagaacgtcctgcacctgtgctgcaagaagctgaagatctttgccatgcctatgcaggacatcaagatgatcctgaagatggtgcagctggacagcatcgaggacctggaagtgacctgtacctggaagctgcccacactggccaagttctttagctacctgggccagatgatcaacctgcggagactgctgctgagccacatccacgccagctcctacatcagccccgagaaagaggaacagtacatctcccagttcacctctcagtttctgagcctgcagtgtctgcaggccctgtacgtggacagcctgttcttcctgagaggcaggctggaccagctgctgagacacgtgatgaaccctctggaaaccctgagcatcaccaactgcagactgctggaaggcgacgtgatgcacctgtctcagagcccatctgtgtcccagctgagcgtgctgtctctgtctggcgtgatgctgaccgatgtgtcccctgaacctctgcaggcactgctgaaaaaggccagcgccactctgcaggacctggtgtttgatgagtgcggcatcatggacgaccagctgtttgccctgctgccaagcctgagccactgtagccaactgaccacactgagcttctacggcaacagcatctacatctctgccctgcagagcctcctgcagcacctgatcggactgagcaatctgacccacgtgctgtacccagtgctgctcgagagctacgaggacatccacgtgaccctgcaccaagagagactggcctatctgcatgcccggctgagagaactgctgtgcgaactgggcagacccagcatggtttggctgagcgctaatctgtgccctcactgcggcgacagaaccttctacgaccccaagctgatcatgtgcccctgcttcatgcccaaccggggcagaaagagaagaagctctagccctggcacagagagcgccggaaagtccctgcagtacagagtggatcatctgctgagcgccgtggaaaacgaactgcaggccggatctgagaagggcgatcctacagagcacgagctgagagtcggcctggaagagtctgagctgtggctgcggttcaaagaactgaccaacgagatgatcgtcaccaagaacggcagacggatgttccccgtgctgaaagtgaacgtgtccggactggaccccaacgccatgtatagctttctgctggacttcgtggtggccgacaaccacagatggaaatacgtgaacggcgagtgggtgccaggcggaaaacctcaactgcaagcccctagctgcgtgtacattcaccctgacagccccaatttcggcgcccactggatgaaggcccctgtgtcctttagcaaagtcaagctgaccaacaagctgaacggcggaggccagatcatgctgaactccctgcacaaatacgagcccagaatccacatcgtcagagtcggcggaccccagagaatgatcaccagccactgcttccccgagacacagtttatcgccgtgaccgcctaccagaacgaggaaatcacaaccctgaagatcaagtacaaccccttcgccaaggccttcctggacgccaaagagcggagcgaccacaaagaaatgatcaaagagcccggcgactcccagcagccaggctattctcaatggggatggctgctgccaggcaccagcacattgtgccctccagccaatcctcacagccagtttggaggcgctctgtccctgagcagcacacacagctacgacagataccccacactgcggagccacagaagcagcccctatccttctccttacgctcaccggaacaacagccccacctacagcgataatagccccgcctgtctgagcatgctgcagtcccacgataattggagcagcctgcggatgcctgctcacccttctatgctgcccgtgtctcacaacgcctctccacctacaagcagctctcagtaccccagcctttggagcgtgtccaatggcgctgtgacactgggatctcaggccgctgctgtgtctaatggactgggagcccagttcttcagaggcagccctgctcactacacccctctgacacatcctgtgtcagccccttctagcagcggcttccctatgtacaaaggcgccgctgccgccaccgatatcgtggattctcagtacgatgccgccgctcagggccacctgattgcatcttggacacctgtgtctccaccttccatgtgatga(SEQ ID NO: 65) modPRAME-modTBXTMERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLLKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLTEKVKQKKNVLHLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFFSYLGQMINLRRLLLSHIHASSYISPEKEEQYISQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLLEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLKKASATLQDLVFDECGIMDDQLFALLPSLSHCSQLTTLSFYGNSIYISALQSLLQHLIGLSNLTHVLYPVLLESYEDIHVTLHQERLAYLHARLRELLCELGRPSMVWLSANLCPHCGDRTFYDPKLIMCPCFMPNRGRKRRSSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTEHELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFWADNHRWKYVNGEWVPGGKPQLQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITTLKIKYNPFAKAFLDAKERSDHKEMIKEPGDSQQPGYSQWGWLLPGTSTLCPPANPHSQFGGALSLSSTHSYDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLRMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTLGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGFPMYKGAAAATDIVDSQYDAAAQGHLIASWTPVSPPSM (SEQ ID NO: 66) HPV16/18 E6/E7atgcaccagaaacggaccgccatgtttcaggaccctcaagagaggcccagaaagctgcctcacctgtgtaccgagctgcagaccaccatccacgacatcatcctggaatgcgtgtactgcaagcagcagctcctgcggagagaggtgtacgatttcgccttccgggacctgtgcatcgtgtacagagatggcaacccctacgccgtgtgcaacaagtgcctgaagttctacagcaagatcagcgagtaccgctactactgctacagcgtgtacggcaccacactggaacagcagtacaacaagcccctgtgcgacctgctgatccggtgcatcaactgccagaaacctctgtgccccgaggaaaagcagcggcacctggacaagaagcagcggttccacaacatcagaggccggtggaccggcagatgcatgagctgttgtcggagcagccggaccagaagagagacacagctgagaggccggaagagaagaagccacggcgatacccctacactgcacgagtacatgctggacctgcagcctgagacaaccgacctgtactgctacgagcagctgaacgacagcagcgaggaagaggacgagattgacggacctgccggacaggccgaacctgatagagcccactacaatatcgtgaccttctgctgcaagtgcaacagcaccctgagactgtgcgtgcagagcacccacgtggacatcagaaccctggaagatctgctgatgggcaccctgggaatcgtgtgccctatctgcagccagaagcctagaggcagaaagcggagaagcgccagattcgacgaccccaccagaaggccttacaagctgcctgatctgtgcactgaactgaacaccagcctgcaggacatcgagattacctgtgtgtattgcaagaccgtgctggaactgaccgaggtgttcgagtttgcctttaaggacctgttcgtggtgtaccgggacagcattcctcacgccgcctgccacaagtgcatcgacttctacagccggatcagagagctgcggcactacagcgattctgtgtacggggacaccctggaaaagctgaccaacaccggcctgtacaacctgctcatcagatgcctgcggtgtcagaagcccctgaatcctgccgagaagctgagacacctgaacgagaagcggagattccacaatatcgccggccactacagaggccagtgccacagctgttgcaaccgggccagacaagagagactgcagagaaggcgggaaacccaagtgcggggcagaaagagaagatctcacggccctaaggccacactgcaggatatcgtgctgcacctggaacctcagaacgagatccccgtggatctgctgtgccatgagcagctgtccgactccaaagaggaaaacgacgaaatcgacggcgtgaaccaccagcatctgcctgccagaagggccgaaccacagagacacaccatgctgtgcatgtgttgcaagtgcgaggcccggattgagctggtggtggaaagctctgccgacgacctgagagccttccagcagctgttcctgaacaccctgagcttcgtgtgtccttggtgcgccagccagcagtgataa (SEQ ID NO: 67) HPV16/18 E6/E7MHQKRTAMFQDPQERPRKLPHLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCNKCLKFYSKISEYRYYCYSVYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLRGRKRRSHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCNSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPRGRKRRSARFDDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQVRGRKRRSHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSKEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ (SEQ ID NO: 68) huPSMAatgtggaatctccttcacgaaaccgactcggctgtggccaccgcgcgccgcccgcgctggctgtgcgctggggcgctggtgctggcgggtggcttctttctcctcggcttcctcttcgggtggtttataaaatcctccaatgaagctactaacattactccaaagcataatatgaaagcatttttggatgaattgaaagctgagaacatcaagaagttcttatataattttacacagataccacatttagcaggaacagaacaaaactttcagcttgcaaagcaaattcaatcccagtggaaagaatttggcctggattctgttgagctagcacattatgatgtcctgttgtcctacccaaataagactcatcccaactacatctcaataattaatgaagatggaaatgagattttcaacacatcattatttgaaccacctcctccaggatatgaaaatgtttcggatattgtaccacctttcagtgctttctctcctcaaggaatgccagagggcgatctagtgtatgttaactatgcacgaactgaagacttctttaaattggaacgggacatgaaaatcaattgctctgggaaaattgtaattgccagatatgggaaagttttcagaggaaataaggttaaaaatgcccagctggcaggggccaaaggagtcattctctactccgaccctgctgactactttgctcctggggtgaagtcctatccagatggttggaatcttcctggaggtggtgtccagcgtggaaatatcctaaatctgaatggtgcaggagaccctctcacaccaggttacccagcaaatgaatatgcttataggcgtggaattgcagaggctgttggtcttccaagtattcctgttcatccaattggatactatgatgcacagaagctcctagaaaaaatgggtggctcagcaccaccagatagcagctggagaggaagtctcaaagtgccctacaatgttggacctggctttactggaaacttttctacacaaaaagtcaagatgcacatccactctaccaatgaagtgacaagaatttacaatgtgataggtactctcagaggagcagtggaaccagacagatatgtcattctgggaggtcaccgggactcatgggtgtttggtggtattgaccctcagagtggagcagctgttgttcatgaaattgtgaggagctttggaacactgaaaaaggaagggtggagacctagaagaacaattttgtttgcaagctgggatgcagaagaatttggtcttcttggttctactgagtgggcagaggagaattcaagactccttcaagagcgtggcgtggcttatattaatgctgactcatctatagaaggaaactacactctgagagttgattgtacaccgctgatgtacagcttggtacacaacctaacaaaagagctgaaaagccctgatgaaggctttgaaggcaaatctctttatgaaagttggactaaaaaaagtccttccccagagttcagtggcatgcccaggataagcaaattgggatctggaaatgattttgaggtgttcttccaacgacttggaattgcttcaggcagagcacggtatactaaaaattgggaaacaaacaaattcagcggctatccactgtatcacagtgtctatgaaacatatgagttggtggaaaagttttatgatccaatgtttaaatatcacctcactgtggcccaggttcgaggagggatggtgtttgagctagccaattccatagtgctcccttttgattgtcgagattatgctgtagttttaagaaagtatgctgacaaaatctacagtatttctatgaaacatccacaggaaatgaagacatacagtgtatcatttgattcactthttctgcagtaaagaattttacagaaattgcttccaagttcagtgagagactccaggactttgacaaaagcaacccaatagtattaagaatgatgaatgatcaactcatgtttctggaaagagcatttattgatccattagggttaccagacaggcctttttataggcatgtcatctatgctccaagcagccacaacaagtatgcaggggagtcattcccaggaatttatgatgctctgtttgatattgaaagcaaagtggacccttccaaggcctggggagaagtgaagagacagatttatgttgcagccttcacagtgcaggcagctgcagagactttgagtgaagtagcctaa (SEQ ID NO: 69) huPSMAMWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLIVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA (SEQ ID NO: 70) CD276 shRNAccggtgctggagaaagatcaaacagctcgagctgtttgatctttctccagcatttttt (SEQ ID NO: 71)modMAGEA1atgtctctcgaacagagaagcctgcactgcaagcccgaggaagctctggaagctcagcaagaggctctgggccttgtgtgtgttcaggccgctgccagcagcttttctcctctggtgctgggcacactggaagaggtgccaacagccggctctaccgatcctcctcaatctcctcaaggcgccagcgcctttcctaccaccatcaacttcacccggcagagacagcctagcgagggctctagctctcacgaggaaaagggccctagcaccagctgcatcctggaaagcctgttccgggccgtgatcacaaagaaagtggccgacctcgtgggcttcctgctgctgaagtacagagccagagaacccgtgaccaaggccgagatgctggaaagcgtgatcaagaactacaagcactgcttcagcgagatcttcggcaaggccagcgagtctctgcagctcgtgtttggcatcgacgtgaaagaggccgatcctaccggccacagctacgtgttcgtgacatgtctgggcctgagctacgatggcctgctgggcgacaatcagattatgctgaaaaccggcttcctgatcatcgtgctggtcatgatcgccatggaaggctctcacgcccctaaagaggaaatctgggaagaactgagcgtgatggaagtgtacgacggcagagagcatagcgcctacggcgagcctagaaaactgctgacccaggacctggtgcaagagaagtacctcgagtacagacaggtgcccgacagcgaccctgccagatacgaatttctgtggggccctagagcactggccgagacaagctatgtgaaggtgctggaatacgtcatcaaggtgtccgccagagtgtgcttcttcttcccatctctgcgggaagccgctctgcgcgaagaggaagaaggcgtc (SEQ ID NO: 72) modMAGEA1MSLEQRSLHCKPEEALEAQQEALGLVCVQAAASSFSPLVLGTLEEVPTAGSTDPPQSPQGASAFPTTINFTRQRQPSEGSSSHEEKGPSTSCILESLFRAVITKKVADLVGFLLLKYRAREPVTKAEMLESVIKNYKHCFSEIFGKASESLQLVFGIDVKEADPTGHSYVFVTCLGLSYDGLLGDNQIMLKTGFLIIVLVMIAMEGSHAPKEEIWEELSVMEVYDGREHSAYGEPRKLLTQDLVQEKYLEYRQVPDSDPARYEFLWGPRALAETSYVKVLEYVIKVSARVCFFFPSLREAALREEEEGV (SEQ ID NO: 73) EGFRvIIIctggaagagaaaaagggcaactacgtggtcaccgaccactgc (SEQ ID NO: 74) EGFRvIIILEEKKGNYVVTDHC (SEQ ID NO: 75) hCMV pp65gagtctagaggcagacggtgccctgagatgattagcgtgctgggccctatctctggccacgtgctgaaggccgtgttcagcagaggcgatacacctgtgctgccccacgagacaagactgctgcagacaggcatccatgtgcgggtgtcacagccaagcctgatcctggtgtctcagtacacccctgacagcaccccttgtcacagaggcgacaaccagctccaggtgcagcacacctactttaccggcagcgaggtggaaaacgtgtccgtgaacgtgcacaatcccaccggcagatccatctgtcccagccaagagcctatgagcatctacgtgtacgccctgcctctgaagatgctgaacatccccagcatcaatgtgcatcactacccctctgccgccgagcggaaacacagacatctgcctgtggccgatgccgtgattcacgcctctggaaagcagatgtggcaggccagactgacagtgtccggactggcttggaccagacagcagaaccagtggaaagaacccgacgtgtactacacctccgccttcgtgttccccacaaaggacgtggccctgagacacgttgtgtgcgcccatgaactcgtgtgcagcatggaaaacacccgggccaccaagatgcaagtgatcggcgaccagtacgtgaaggtgtacctggaatccttctgcgaggacgtgccaagcggcaagctgttcatgcacgtgaccctgggctccgatgtggaagaggacctgaccatgaccagaaatccccagcctttcatgcggcctcacgagagaaatggcttcaccgtgctgtgccccaagaacatgatcatcaagcccggcaagatcagccacatcatgctggatgtggccttcaccagccacgagcacttcggactgctgtgtcctaagagcatccccggcctgagcatcagcggcaacctgctgatgaatggccagcagatcttcctggaagtgcaggccattcgggaaaccgtggaactgagacagtacgaccctgtggctgccctgttcttcttcgacatcgatctgctgctccagagaggccctcagtacagcgagcacccaacctttaccagccagtacagaatccagggcaagctggaatatcggcacacctgggatagacacgatgagggtgctgcacagggcgacgatgatgtgtggacaagcggcagcgatagcgacgaggaactggtcaccaccgagagaaagacccctagagttacaggcggaggcgcaatggctggcgcttctacatctgccggacgcaagagaaagagcgcctcttctgccaccgcctgtacaagcggcgtgatgacaagaggcaggctgaaagccgagagcacagtggcccctgaggaagatacagacgaggacagcgacaacgagattcacaaccccgccgtgtttacctggcctccttggcaggctggcattctggctagaaacctggtgcctatggtggccacagtgcagggccagaacctgaagtaccaagagttcttctgggacgccaacgacatctaccggatcttcgccgaactggaaggcgtgtggcaaccagccgctcagcccaaaagacgcagacacagacaggacgctctgcccggaccttgtattgccagcacacccaagaaacaccggggc (SEQ ID NO: 76) hCMV pp65ESRGRRCPEMISVLGPISGHVLKAVFSRGDTPVLPHETRLLQTGIHVRVSQPSLILVSQYTPDSTPCHRGDNQLQVQHTYFTGSEVENVSVNVHNPTGRSICPSQEPMSIYVYALPLKMLNIPSINVHHYPSAAERKHRHLPVADAVIHASGKQMWQARLTVSGLAWTRQQNQWKEPDVYYTSAFVFPTKDVALRHVVCAHELVCSMENTRATKMQVIGDQYVKVYLESFCEDVPSGKLFMHVTLGSDVEEDLTMTRNPQPFMRPHERNGFTVLCPKNMIIKPGKISHIMLDVAFTSHEHFGLLCPKSIPGLSISGNLLMNGQQIFLEVQAIRETVELRQYDPVAALFFFDIDLLLQRGPQYSEHPTFTSQYRIQGKLEYRHTWDRHDEGAAQGDDDVWTSGSDSDEELVTTERKTPRVTGGGAMAGASTSAGRKRKSASSATACTSGVMTRGRLKAESTVAPEEDTDEDSDNEIHNPAVFTWPPWQAGILARNLVPMVATVQGQNLKYQEFFWDANDIYRIFAELEGVWQPAAQPKRRRHRQDALPGPCIASTPKKHRG (SEQ ID NO: 77) modTBXTatgtctagccctggaacagagtctgccggcaagagcctgcagtacagagtggaccatctgctgagcgccgtggaaaatgaactgcaggccggaagcgagaagggcgatcctacagagcacgagctgagagtcggcctggaagagtctgagctgtggctgcggttcaaagaactgaccaacgagatgatcgtgaccaagaacggcagacggatgttccccgtgctgaaagtgaacgtgtccggactggaccccaacgccatgtacagctttctgctggacttcgtggtggccgacaaccacagatggaaatacgtgaacggcgagtgggtgccaggcggaaaacctcaactgcaagcccctagctgcgtgtacattcaccctgacagccccaatttcggcgcccactggatgaaggcccctgtgtccttcagcaaagtgaagctgaccaacaagctgaacggcggaggccagatcatgctgaacagcctgcacaaatacgagcccagaatccacatcgtcagagtcggcggaccccagagaatgatcaccagccactgcttccccgagacacagtttatcgccgtgaccgcctaccagaacgaggaaatcaccacactgaagatcaagtacaaccccttcgccaaggccttcctggacgccaaagagcggagcgaccacaaagagatgatcaaagagcccggcgacagccagcagccaggctattctcaatggggatggctgctgccaggcaccagcacattgtgccctccagccaatcctcacagccagtttggaggcgccctgagcctgtctagcacccacagctacgacagataccccacactgcggagccacagaagcagcccctatccttctccttacgctcaccggaacaacagccccacctacagcgataatagccccgcctgtctgagcatgctgcagtcccacgataactggtccagcctgagaatgcctgctcacccttccatgctgcccgtgtctcacaatgcctctccacctaccagcagctctcagtaccctagcctttggagcgtgtccaatggcgccgtgacactgggatctcaggcagccgctgtgtctaatggactgggagcccagttcttcagaggcagccctgctcactacacccctctgacacatcctgtgtctgcccctagcagcagcggcttccctatgtataagggcgctgccgccgctaccgacatcgtggattctcagtatgatgccgccgcacagggacacctgatcgcctcttggacacctgtgtctccaccttccatg (SEQ ID NO: 78)modTBXTMSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTEHELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVVADNHRWKYVNGEWVPGGKPQLQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITTLKIKYNPFAKAFLDAKERSDHKEMIKEPGDSQQPGYSQWGWLLPGTSTLCPPANPHSQFGGALSLSSTHSYDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLRMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTLGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGFPMYKGAAAATDIVDSQYDAAAQGHLIASWTPVSPPSM (SEQ ID NO: 79) modWTIgacttcctgctgctgcagaaccctgcctctacctgtgtgcctgaaccagcctctcagcacaccctgagatctggccctggatgtctccagcagcctgaacagcagggcgttagagatcctggcggaatctgggccaaactgggagctgccgaagcctctgccgaatgtctgcagggcagaagaagcagaggcgccagcggatctgaacctcaccagatgggaagcgacgtgcacgacctgaatgctctgttgcctgccgtgccatctcttggcggaggcggaggatgtgctttgcctgtttctggtgctgcccagtgggctcccgtgctggattttgctcctcctggcgcttctgcctatggctctcttggaggacctgctcctccaccagctccacctccaccgccgcctccaccacctcacagctttatcaagcaagagccctcctggggcggagccgagcctcacgaaaaacagtgtctgagcgccttcaccgtgcactttttcggccagtttaccggcaccgtgggcgcctgtagatacggcccttttggaccaccaccacctagccaggcttctagcggacaggccagaatgttccccaacgctccttacctgcctagctgcctggaaagccagcctaccatcagaaaccagggcttcagcaccgtgaccttcgacggcatgcctagctatggccacacaccatctcaccacgccgctcagttccccaatcacagcttcaagcacgaggaccctatgggccagcagggatctctgggagagcagcagtatagcgtgccacctcctgtgtacggctgtcacacccctaccgatagctgcacaggcaatcaggctctgctgctgaggatgcctttcagcagcgacaacctgtaccagatgacaagccagctggaatgcatgatttggaaccagatgaacctgggcgccactctgaaaggcgtggccgctggatctagcagctccgtgaaatggacagccggccagagcaatcactccaccggctacgagagcgacaatcacaccatgcctatcctgtgtggggcccagtaccggattcacacacacggcgtgttcaggggcattcaggatgtgcgaagagtgcctggcgtggcccctacacttgtgggatctgccagcgaaaccagcgagaagcaccccttcatgtgcgcctatccaggctgcaacaagcggtacttcaagctgagccacctgaagatgcacagccggaagcacacaggcgagaagctgtaccagtgcgacttcaaggactgcgagcggagattcagctgcagcgaccagctgaagagacaccagagaaggcacaccggcgtgaagccctttcagtgcaagacctgccagcggaccttctcctggtccaaccacctgaaaacccacacaagaacccacaccggcaagaccatcgagaagcccttcagctgtagatggcccagctgccagaagaagttcgcccggtctaacgagctggtgcatcaccacaacatgcaccagaggaacatgaccaaactgcagctggtgctg (SEQ ID NO: 80)modWT1 DFLLLQNPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAECLQGRRSRGASGSEPHQMGSDVHDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEKQCLSAFTVHFFGQFTGTVGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPTIRNQGFSTVTFDGMPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGNQALLLRMPFSSDNLYQMTSQLECMIWNQMNLGATLKGVAAGSSSSVKWTAGQSNHSTGYESDNHTMPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVGSASETSEKHPFMCAYPGCNKRYFKLSHLKMHSRKHTGEKLYQCDFKDCERRFSCSDQLKRHQRRHTGVKPFQCKTCQRTFSWSNHLKTHTRTHTGKTIEKPFSCRWPSCQKKFARSNELVHHHNMHQRNMTKLQLVL (SEQ ID NO: 81) KRAS G12D mutationaccgagtacaagctggtggttgttggagccgatggcgtgggaaagagcgccctgacaattcagctgatccagaaccacttcgtg (SEQ ID NO: 82) KRAS G12D mutation TEYKLVVVGADGVGKSALTIQLIQNHFV (SEQ ID NO: 83)KRAS G12V mutationacagagtataagctcgtggtcgtgggcgctgtcggagtgggaaaatctgccctgaccatccaactcattcagaatcactttgtg (SEQ ID NO: 84)KRAS G12V mutation TEYKLVVVGAVGVGKSALTIQLIQNHFV (SEQ ID NO: 85)modMAGEC2cctcctgtgcctggcgtgcccttcagaaacgtggacaacgatagcctgaccagcgtggaactggaagattgggtcgacgcccagcatccvtaccgacgaggaagaggaagaagccagctctgccagcagcaccctgtacctggtgtttagccccagcagcttctccaccagctctagcctgattctcggaggccccgaagaagaagaggtcccaagcggcgtgatccccaatctgacagagagcatcccaagcagccctccacagggaccaccacaaggaccttctcagagccctctgagcagctgttgcagcagtttcctgtggtccagcttcagcgaggaaagcagctcccagaaaggcgaggataccggcacttgtcagggcctgccagatagcgagagcagcttcacctacacactggacgagaaggtggccaagctggtcgagttcctgctgctgaagtacgaggccgaggaacctgtgacagaggccgagatgctgatgatcgtcatcaagtataaggactacttccccgtgatcctgaagcgggccagagaattcatggaactgctgttcggactggccctgatcgaagtgggccccgatcacttctgcgtgttcgctaacacagtgggcctgaccgatgagggctccgatgatgagggaatgcccgagaactccctgctgatcatcatcctgagcgtcatcttcatcaagggcaactgcgcctccgaggaagtgatctgggaagtcctgaatgccgtgggcgtttacgccggcagagaacactttgtgtacggcaagccccgcgagctgctgaccaatgtttgggtgcagggccactacctggaatactgggaagtgcctcactctagccctctgtactacgagtttctgtggggccctagagcacacagcgagtccatcaagaaaaaggtgctggaattcctggccaaactgaacaataccgtgcctagcttcttcccgtcctggtacaaggatgccctgaaggacgtggaagagagagtgcaggccaccatcgacaccgccgatgatgctacagtgatggccagcgagagcctgagcgtgatgagcagcaacgtgtcctttagcgag (SEQ ID NO: 86)modMAGEC2PPVPGVPFRNVDNDSLTSVELEDWVDAQHPTDEEEEEASSASSTLYLVFSPSSFSTSSSLILGGPEEEEVPSGVIPNLTESIPSSPPQGPPQGPSQSPLSSCCSSFLWSSFSEESSSQKGEDTGTCQGLPDSESSFTYTLDEKVAKLVEFLLLKYEAEEPVTEAEMLMIVIKYKDYFPVILKRAREFMELLFGLALIEVGPDHFCVFANTVGLTDEGSDDEGMPENSLLIIILSVIFIKGNCASEEVIWEVLNAVGVYAGREHFVYGKPRELLTNVWVQGHYLEYWEVPHSSPLYYEFLWGPRAHSESIKKKVLEFLAKLNNTVPSFFPSWYKDALKDVEERVQATIDTADDATVMASESLSVMSSNVSFSE (SEQ ID NO: 87) modTDGF1gactgcagaaagatggcccggttcagctactccgtgatctggatcatggccatctccaaggccttcgagctgagactggttgccggactgggccaccaagagtttgccagacctagctggggctatctggccttccgggacgatagcatctggccccaagaggaacctgccatcagacccagatctagccagcgggtgccacctatggaaatccagcacagcaaagaactgaaccggacctgctgcctgaacggcagaacctgtatgctgggcagcttctgcgcctgtcctcctagcttctacggccggaattgcgagcacgacgtgcggaaagaaaactgcggcagcgtgccacacgatacctggctgcctaagaaatgcagcctgtgcaagtgttggcacggccagctgcggtgtttccccagagcttttctgcccgtgtgtgacggcctggtcatggatgaacacctggtggccagcagaacccctgagcttcctccaagcgccaggaccaccacctttatgctcgtgggcatctgcctgagcatccagagctactac (SEQ ID NO: 88) modTDGF1DCRKMARFSYSVIWIMAISKAFELRLVAGLGHQEFARPSWGYLAFRDDSIWPQEEPAIRPRSSQRVPPMEIQHSKELNRTCCLNGRTCMLGSFCACPPSFYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPRAFLPVCDGLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYY (SEQ IDNO: 89) modPSMA in modPSMA_TDGF1ggatccgccaccatgtggaatctgctgcacgagacagatagcgccgtggctaccgttagaaggcccagatggctttgtgctggcgctctggttctggctggcggcttttttctgctgggcttcctgttcggctggttcatcaagagcagcaacgaggccaccaacatcacccctaagcacaacatgaaggcctttctggacgagctgaaggccgagaatatcaagaagttcctgtacaacttcacgcacatccctcacctggccggcaccgagcagaattttcagctggccaagcagatccagagccagtggaaagagttcggcctggactctgtggaactggcccactacgatgtgctgctgagctaccccaacaagacacaccccaactacatcagcatcatcaacgaggacggcaacgagatcttcaacaccagcctgttcgagcctccacctcctggctacgagaacgtgtccgatatcgtgcctccattcagcgctttcagcccacagcggatgcctgagggctacctggtgtacgtgaactacgccagaaccgaggacttcttcaagctggaatgggacatgaagatcagctgcagcggcaagatcgtgatcgcccggtacagaaaggtgttccgcgagaacaaagtgaagaacgcccagctggcaggcgccaaaggcgtgatcctgtatagcgaccccgccgactattttgcccctggcgtgaagtcttaccccgacggctggaattttcctggcggcggagtgcagcggcggaacatccttaatcttaacggcgctggcgaccctctgacacctggctatcctgccaatgagtacgcctacagacacggaattgccgaggctgtgggcctgccttctattcctgtgcaccctgtgcggtactacgacgcccagaaactgctggaaaagatgggcggaagcgcccctcctgactcttcttggagaggctctctgaaggtgccctacaatgtcggcccaggcttcaccggcaacttcagcacccagaaagtgaaaatgcacatccacagcaccaacgaagtgacccggatctacaacgtgatcggcacactgagaggcgccgtggaacccgacaaatacgtgatcctcggcggccacagagacagctgggtgttcggaggaatcgaccctcaatctggcgccgctgtggtgtatgagatcgtgcggtctttcggcaccctgaagaaagaaggatggcggcccagacggaccatcctgtttgcctcttgggacgccgaggaatttggcctgctgggatctacagagtgggccgaagagaacagcagactgctgcaagaaagaggcgtggcctacatcaacgccgacagcagcatcgagggcaactacaccctgcggatcgattgcacccctctgatgtacagcctggtgcacaacctgaccaaagagctgaagtcccctgacgagggctttgagggcaagagcctgtacaagagctggaccaagaagtccccatctcctgagttcagcggcatgcccagaatctctaagctggaaagcggcaacaacttcgaggtgttcttccagcggctgggaatcgcctctggaatcgccagatacaccaagaactgggagacaaacaagttctccggctatcccctgtaccacagcgtgtacgagacatacgagctggtggaaaagttctacgaccccatgttcaagtaccacctgacagtggcccaagtgcgcggaggcatggtgttcgaactggccaatagcatcgtgctgcccttcaactgcagagactacgccgtggtgctgcggaagtacgccgacaagatctacagcatcagcatgaagcacccgcaagagatgaagacctacagcgtgtccttcgactccctgttcttcgccgtgaagaacttcaccaagatcgccagcaagttcagcgagcggctgcaggacttcgacaagagcaaccctatcgtgctgaggatgatgaacgaccagctgatgttcctggaacgggccttcatcaaccctctgggactgcccgacagacccttctacaggcacgtgatctgtgcccctagcagccacaacaaatacgccggcgagagcttccccggcatctacgatgccctgttcgacatcgagagcaacgtgaaccctagcaaggcctggggcgaagtg aagagacagatctacgtggccgcattcacagtgcaggccgctgccgaaacactgtctgaagtggccagaggc (SEQ ID NO: 90)modWT1 in modWT1_FBPatggactttctgctgctgcagaaccctgccagcacctgtgttccagaacctgcctctcagcacaccctgagatctggccctggatgtctccagcagcctgaacagcagggcgttagagatcctggcggaatctgggccaaactgggagccgctgaagcctctgccgaatgtctgcagggcagaagaagcagaggcgccagcggatctgaacctcaccagatgggaagcgacgtgcacgacctgaatgctctgctgcctgccgtgccatctcttggcggaggcggaggatgtgctttgcctgtttctggtgctgcccagtgggctcccgtgctggattttgctcctcctggcgcttctgcctatggctctcttggaggacctgctcctccaccagctccacctccaccgccgcctccaccacctcacagctttatcaagcaagagccctcctggggcggagccgagcctcacgaaaaacagtgtctgagcgccttcaccgtgcactttttcggccagtttaccggcacagtgggcgcctgtagatacggcccttttggaccaccaccacctagccaggctagctctggacaggccagaatgttccccaacgctccctacctgcctagctgcctggaaagccagcctaccatcagaaaccagggcttcagcaccgtgaccttcgacggcatgcctagctatggccacacaccatctcaccacgccgctcagttccccaatcacagcttcaagcacgaggaccctatgggccagcagggatctctgggagagcagcagtatagcgtgccacctcctgtgtacggctgtcacacccctaccgatagctgcacaggcaatcaggccctgctgctgaggatgcccttcagcagcgacaacctgtaccagatgacaagccagctggaatgcatgatctggaaccagatgaacctgggcgccacactgaaaggcgtggccgctggatctagcagcagcgtgaaatggacagccggccagagcaatcactccaccggctacgagtccgacaaccacaccatgcctattctgtgcggagcccagtacagaatccacacacacggcgtgttccggggcattcaggatgtgcgaagagtgcctggcgtggcccctacacttgtgggatctgcctctgagacaagcgagaagcaccccttcatgtgcgcctatcctggctgcaacaagcggtacttcaagctgagccacctgaagatgcacagccggaagcacacaggcgagaagctgtaccagtgcgacttcaaggactgcgagcggagattcagctgcagcgaccagctgaagagacaccagagaaggcacaccggcgtgaagcccttccagtgcaagacctgccagcggacctttagctggtccaaccacctgaaaacccacacaagaacccacaccggcaagaccatcgagaagcctttcagctgtagatggcccagctgccagaagaagttcgcccggtctaacgagctggtgcatcaccacaacatgcaccagaggaacatgaccaaactgcagctggtgctg (SEQ ID NO: 91)modFBPgcccagagaatgaccacacagttgctgctgctcctcgtgtgggttgccgttgtgggagaagtgcagaccagaatcgcctgggccagaaccgagctgctgaacgtgtgcatgaacgccaagcaccacaagaagaagcccgatcctgaggacaagctgcacgagcagtgtcggccttggagaaagaacgcctgctgtagcaccaacaccagccaagaggcccacaagaacgtgtcctacctgtaccggttcaactggaaccactgcggcgagatgacacccgcctgcaagagacacttcatccaggatacctgcctgtacgagtgcagccccaatctcggcccctggattcagcaagtggaccagagctggcggaaagaactggtcctgaatgtgcccctgtgcaaagaggattgcgagcagtggtgggaagattgcagaaccagctacacatgcaagagcaactggcacaaaggctggaactggaccagcggcttcaacaagtgtgccgtgggagctgcctgtcagcctttccacttctactttcacacacccaccgtgctgtgcaacaagatctggacccacagctacaaggtgtccaactacagcagaggcagcggccggtgtatccagatgtggttcgatcccgccaagggcaaccccaatgaggaagtggccagattctacgccgctgccatgtctggtgcaggaccttgggctgcttggccctttctgctttcactggccctgatgctgctgtggctgctgagc (SEQ ID NO: 92)modFBP AQRMTTQLLLLLVWVAVVGEVQTRIAWARTELLNVCMNAKHHKKKPDPEDKLHEQCRPWRKNACCSTNTSQEAHKNVSYLYRFNWNHCGEMTPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKELVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFHTPTVLCNKIWTHSYKVSNYSRGSGRCIQMWFDPAKGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS (SEQ ID NO: 93) modFSHRatggctctgctgctggtttctctgctggccctgctgtctctcggctctggatgtcaccacagaatctgccactgcagcaaccgggtgttcctgtgccagaaaagcaaagtgaccgagatcctgagcgacctgcagcggaatgccatcgagctgagattcgtgctgaccaagctgcaagtgatccagaagggcgccttcagcggcttcggcgacctggaaaagatcgagatcagccagaacaacgtgctggaagtgatcgaggcccacgtgttcagcaacctgcctaagctgcacgagatcagaatcgagaaggccaacaacctgctgtacatcaaccccgaggccttccagaacttccccaacctgcagtacctgctgatctccaacaccggcatcaaacatctgcccgacgtgcacaagatccacagcctgcagaaggtgctgctggacatccaggacaacatcaacatccacacaatcgagcggaactacttcctgggcctgagcttcgagagcgtgatcctgtggctgaacaagaacggcatccaagagatccacaactgcgccttcaatggcacccagctggacgagctgaacctgtccgacaacaacaatctggaagaactgcccaacgacgtgttccacagagccagcggacctgtgatcctggacatcagcagaaccagaatccactctctgcccagctacggcctggaaaacctgaagaagctgcgggccagaagcacctacaatctgaaaaagctgcctacgctggaaaccctggtggccctgatggaagccagcctgacataccctagccactgctgcgcctttgccaactggcggagacagatctctgagctgcaccccatctgcaacaagagcatcctgcggcaagaggtggactacatgacacaggccagaggccagagattcagcctggccgaggataacgagagcagctacagcagaggcttcgacatgacctacaccgagttcgactacgacctgtgcaacaaggtggtggacgtgacatgcagccccaagcctgatgccttcaatccctgcgaggacatcatgggctacaacatcctgagagtgctgatctggttcatcagcatcctggccatcaccgagaacatcatcgtgctggtcatcctgaccaccagccagtacaagctgaccgtgcctatgttcctgatgtgcaacctggccttcgccgatctgtgcatcggcatctacctgctgctgatcgccagcgtggacattcacaccaagagccagtaccacaactacgccatcgactggcagacaggcgccggatgtgatgccgccggattctttacagtgttcgccagcgagctgtccgtgtacaccctgacagctatcaccctggaacggtggcacaccatcacacacgctatgcagctggactgcaaagtgcacctgagacacagcgcctccgtgatggttatgggctggatcttcgccttcgctgccgctctgttccccatctttggcatcagctcctacatgaaggtgtccatctatctgcccatggacatcgacagccctctgagccagctgtacgtgatgagtctgctggtgctgaatgtgctggcctttgtggtcatctgcggctgctacatctatatctacctgacagtgcggaaccccaacatcgtgtccagctccagcgacacccggatcgctaagagaatggccatgctgatcttcaccgactttctgtgcatggcccctatcagcctgttcgccattagcgctagcctgaaggtgcccctgatcaccgtgtccaaggccaagattctgctggtcctgttctaccccatcaacagctgcgccaatcctttcctgtacgccatcttcaccaagaacttcaggcggaacttcttcatcctgctgagcaagcggggctgttacaagatgcaggcccagatctaccggaccgagacactgtccaccgtgcacaacacacaccccagaaacggccactgtagcagcgcccctagagtgacaaatggctccacctacatcctggtgccactgagccatctggcccagaac (SEQ ID NO: 94) modFSHRMALLLVSLLALLSLGSGCHHRICHCSNRVFLCQKSKVTEILSDLQRNAIELRFVLTKLQVIQKGAFSGFGDLEKIEISQNNVLEVIEAHVFSNLPKLHEIRIEKANNLLYINPEAFQNFPNLQYLLISNTGIKHLPDVHKIHSLQKVLLDIQDNINIHTIERNYFLGLSFESVILWLNKNGIQEIHNCAFNGTQLDELNLSDNNNLEELPNDVFHRASGPVILDISRTRIHSLPSYGLENLKKLRARSTYNLKKLPTLETLVALMEASLTYPSHCCAFANWRRQISELHPICNKSILRQEVDYMTQARGQRFSLAEDNESSYSRGFDMTYTEFDYDLCNKVVDVTCSPKPDAFNPCEDIMGYNILRVLIWFISILAITENIIVLVILTTSQYKLTVPMFLMCNLAFADLCIGIYLLLIASVDIHTKSQYHNYAIDWQTGAGCDAAGFFTVFASELSVYTLTAITLERWHTITHAMQLDCKVHLRHSASVMVMGWIFAFAAALFPIFGISSYMKVSIYLPMDIDSPLSQLYVMSLLVLNVLAFVVICGCYIYIYLTVRNPNIVSSSSDTRIAKRMAMLIFTDFLCMAPISLFAISASLKVPLITVSKAKILLVLFYPINSCANPFLYAIFTKNFRRNFFILLSKRGCYKMQAQIYRTETLSTVHNTHPRNGHCSSAPRVTNGSTYILVPLSHLAQN (SEQ ID NO: 95) modMAGEA10cccagggctcccaagagacagagatgcatgcccgaagaggacctgcagagccagagcgaaacacagggactcgaaggtgctcaggctcctctggccgtggaagaagatgccagcagctctaccagcacctccagcagcttccctagcagctttccattcagctcctctagctctagcagcagctgttaccctctgatccccagcacacccgagaaggtgttcgccgacgacgagacacctaatccactgcagtctgcccagatcgcctgcagcagtacactggtggttgctagcctgcctctggaccagtctgatgagggaagcagcagccagaaagaggaaagccctagcacactccaggtgctgcccgatagcgagagcctgcctagaagcgagatctacaagaaaatgaccgacctggtgcagttcctcctgttcaagtaccagatgaaggaacccatcaccaaggccgaaatcctggaaagcgtgatcagaaactacgaggaccactttccactgctgttcagcgaggccagcgagtgcatgctgctcgtgtttagcatcgacgtgaagaaggtggaccccaccggccacagctttgtgctggttacaagcctgggactgacctacgacggcatgctgtccgatgtgcagagcatgcctaagaccggcatcctgatcctgattctgagcatcgtgttcatcgagggctactgcacccctgaggaagtgatttgggaagccctgaacatgatgggcctgtacgatggcatggaacacctgatctacggcgagcccagaaaactgctgacccaggactgggtgcaagagaactacctggaataccggcagatgcccggcagcgatcctgccagatatgagtttctgtggggccctagagcacatgccgagatccggaagatgagcctgctgaagttcctggccaaagtgaacggcagcgacccaatcagcttcccactttggtacgaagaggccctgaaggacgaggaagagagagcccaggatagaatcgccaccaccgacgacacaacagccatggcctctgcctcttctagcgccaccggcagctttagctaccccgag (SEQ ID NO: 96) modMAGEA10PRAPKRQRCMPEEDLQSQSETQGLEGAQAPLAVEEDASSSTSTSSSFPSSFPFSSSSSSSSCYPLIPSTPEKVFADDETPNPLQSAQIACSSTLVVASLPLDQSDEGSSSQKEESPSTLQVLPDSESLPRSEIYKKMTDLVQFLLFKYQMKEPITKAEILESVIRNYEDHFPLLFSEASECMLLVFSIDVKKVDPTGHSFVLVTSLGLTYDGMLSDVQSMPKTGILILILSIVFIEGYCTPEEVIWEALNMMGLYDGMEHLIYGEPRKLLTQDWVQENYLEYRQMPGSDPARYEFLWGPRAHAEIRKMSLLKFLAKVNGSDPISFPLWYEEALKDEEERAQDRIATTDDTTAMASASSSATGSFSYPE (SEQ ID NO: 97) modPRAMEatggaaagaagaaggctctggggcagcatccagagccggtacatcagcatgagcgtgtggacaagccctcggagactggtggaactggctggacagagcctgctgaaggatgaggccctggccattgctgctctggaactgctgcctagagagctgttccctcctctgttcatggccgccttcgacggcagacacagccagacactgaaagccatggtgcaggcctggcctttcacctgtctgcctctgggagtgctgatgaagggccagcatctgcacctggaaaccttcaaggccgtgctggatggcctggatgtgctgctggctcaagaagtgcggcctcggcgttggaaactgcaggttctggatctgctgaagaacagccaccaggatttctggaccgtttggagcggcaacagagccagcctgtacagctttcctgagcctgaagccgctcagcccatgaccaagaaaagaaaggtggacggcctgagcaccgaggccgagcagccttttattcccgtggaagtgctggtggacctgttcctgaaagaaggcgcctgcgacgagctgttcagctacctgaccgagaaagtgaagcagaagaagaacgtcctgcacctgtgctgcaagaagctgaagatctttgccatgcctatgcaggacatcaagatgatcctgaagatggtgcagctggacagcatcgaggacctggaagtgacctgtacctggaagctgcccacactggccaagttctttagctacctgggccagatgatcaacctgcggagactgctgctgagccacatccacgccagctcctacatcagccccgagaaagaggaacagtacatctcccagttcacctctcagtttctgagcctgcagtgtctgcaggccctgtacgtggacagcctgttcttcctgagaggcaggctggaccagctgctgagacacgtgatgaaccctctggaaaccctgagcatcaccaactgcagactgctggaaggcgacgtgatgcacctgtctcagagcccatctgtgtcccagctgagcgtgctgtctctgtctggcgtgatgctgaccgatgtgtcccctgaacctctgcaggcactgctgaaaaaggccagcgccactctgcaggacctggtgtttgatgagtgcggcatcatggacgaccagctgtttgccctgctgccaagcctgagccactgtagccaactgaccacactgagcttctacggcaacagcatctacatctctgccctgcagagcctcctgcagcacctgatcggactgagcaatctgacccacgtgctgtacccagtgctgctcgagagctacgaggacatccacgtgaccctgcaccaagagagactggcctatctgcatgcccggctgagagaactgctgtgcgaactgggcagacccagcatggtttggctgagcgctaatctgtgccctcactgcggcgacagaaccttctacgaccccaagctgatcatgtgcccctgcttcatgcccaac (SEQ ID NO: 98) modPRAMEMERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLLKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLTEKVKQKKNVLHLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFFSYLGQMINLRRLLLSHIHASSYISPEKEEQYISQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLLEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLKKASATLQDLVFDECGIMDDQLFALLPSLSHCSQLTTLSFYGNSIYISALQSLLQHLIGLSNLTHVLYPVLLESYEDIHVTLHQERLAYLHARLRELLCELGRPSMVWLSANLCPHCGDRTFYDPKLIMCPCFMPN (SEQ ID NO: 99)modTBXT in modPRAME_TBXTtctagccctggcacagagagcgccggaaagtccctgcagtacagagtggatcatctgctgagcgccgtggaaaacgaactgcaggccggatctgagaagggcgatcctacagagcacgagctgagagtcggcctggaagagtctgagctgtggctgcggttcaaagaactgaccaacgagatgatcgtcaccaagaacggcagacggatgttccccgtgctgaaagtgaacgtgtccggactggaccccaacgccatgtatagctttctgctggacttcgtggtggccgacaaccacagatggaaatacgtgaacggcgagtgggtgccaggcggaaaacctcaactgcaagcccctagctgcgtgtacattcaccctgacagccccaatttcggcgcccactggatgaaggcccctgtgtcctttagcaaagtcaagctgaccaacaagctgaacggcggaggccagatcatgctgaactccctgcacaaatacgagcccagaatccacatcgtcagagtcggcggaccccagagaatgatcaccagccactgcttccccgagacacagtttatcgccgtgaccgcctaccagaacgaggaaatcacaaccctgaagatcaagtacaaccccttcgccaaggccttcctggacgccaaagagcggagcgaccacaaagaaatgatcaaagagcccggcgactcccagcagccaggctattctcaatggggatggctgctgccaggcaccagcacattgtgccctccagccaatcctcacagccagtttggaggcgctctgtccctgagcagcacacacagctacgacagataccccacactgcggagccacagaagcagcccctatccttctccttacgctcaccggaacaacagccccacctacagcgataatagccccgcctgtctgagcatgctgcagtcccacgataattggagcagcctgcggatgcctgctcacccttctatgctgcccgtgtctcacaacgcctctccacctacaagcagctctcagtaccccagcctttggagcgtgtccaatggcgctgtgacactgggatctcaggccgctgctgtgtctaatggactgggagcccagttcttcagaggcagccctgctcactacacccctctgacacatcctgtgtcagccccttctagcagcggcttccctatgtacaaaggcgccgctgccgccaccgatatcgtggattctcagtacgatgccgccgctcagggccacctgattgcatcttggacacctgtgtctccaccttccatg (SEQ ID NO: 100)HPV16 E6atgcaccagaaacggaccgccatgtttcaggaccctcaagagaggcccagaaagctgcctcacctgtgtaccgagctgcagaccaccatccacgacatcatcctggaatgcgtgtactgcaagcagcagctcctgcggagagaggtgtacgatttcgccttccgggacctgtgcatcgtgtacagagatggcaacccctacgccgtgtgcaacaagtgcctgaagttctacagcaagatcagcgagtaccgctactactgctacagcgtgtacggcaccacactggaacagcagtacaacaagcccctgtgcgacctgctgatccggtgcatcaactgccagaaacctctgtgccccgaggaaaagcagcggcacctggacaagaagcagcggttccacaacatcagaggccggtggaccggcagatgcatgagctgttgtcggagcagccggaccagaagagagacacagctg (SEQ ID NO: 101) HPV16 E6MHQKRTAMFQDPQERPRKLPHLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCNKCLKFYSKISEYRYYCYSVYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL (SEQ ID NO: 102) HPV16 E7cacggcgatacccctacactgcacgagtacatgctggacctgcagcctgagacaaccgacctgtactgctacgagcagctgaacgacagcagcgaggaagaggacgagattgacggacctgccggacaggccgaacctgatagagcccactacaatatcgtgaccttctgctgcaagtgcaacagcaccctgagactgtgcgtgcagagcacccacgtggacatcagaaccctggaagatctgctgatgggcaccctgggaatcgtgtgccctatctgcagccagaagcct (SEQ ID NO: 103) HPV16 E7HGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCNSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP (SEQ ID NO: 104) HPV18 E6gccagattcgacgaccccaccagaaggccttacaagctgcctgatctgtgcactgaactgaacaccagcctgcaggacatcgagattacctgtgtgtattgcaagaccgtgctggaactgaccgaggtgttcgagtttgcctttaaggacctgttcgtggtgtaccgggacagcattcctcacgccgcctgccacaagtgcatcgacttctacagccggatcagagagctgcggcactacagcgattctgtgtacggggacaccctggaaaagctgaccaacaccggcctgtacaacctgctcatcagatgcctgcggtgtcagaagcccctgaatcctgccgagaagctgagacacctgaacgagaagcggagattccacaatatcgccggccactacagaggccagtgccacagctgttgcaaccgggccagacaagagagactgcagagaaggcgggaaacccaagtg (SEQ ID NO: 105) HPV18 E6ARFDDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV (SEQ ID NO: 106) HPV18 E7cacggccctaaggccacactgcaggatatcgtgctgcacctggaacctcagaacgagatccccgtggatctgctgtgccatgagcagctgtccgactccaaagaggaaaacgacgaaatcgacggcgtgaaccaccagcatctgcctgccagaagggccgaaccacagagacacaccatgctgtgcatgtgttgcaagtgcgaggcccggattgagctggtggtggaaagctctgccgacgacctgagagccttccagcagctgttcctgaacaccctgagcttcgtgtgtccttggtgcgccagccagcag (SEQ ID NO: 107)HPV18 E7HGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSKEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ (SEQ ID NO: 108)modClaudin 18 (CLDN18)gccgtgacagcctgtcagagcctgggctttgtggtgtccctgatcgagatcgtgggcatcattgccgctacctgcatggaccagtggtctacccaggacctgtacaacaaccctgtgaccgccgtgttcaactaccaaggcctgtggcacagctgcatgagagagagcagcggcttcaccgagtgcagaggctacttcaccctgctggaactgcctgccatgctgcaggctgtgcaggcccttatgatcgtgggaattgtgctgggagccatcggcctgctggtgtccattttcgccctgaagtgcatccggatcggcagcatggaagatagcgccaaggccaacatgaccctgaccagcggcatcatgttcatcgtgtccggcctgtgcgccattgctggcgtgtccgtgtttgccaatatgctcgtgaccaacttctggctgagcaccgccaacatgtacaccggcatgggcgagatggtgcagaccgtgcagacacggtacacatttggcgccgctctgtttgtcggatgggttgcaggcggactgacactgattggcggcgtgatgatgtgtatcgcctgcagaggactggcccctgaggaaacaaactacaaggccgtgtactaccacgcctccggacacagcgtggcatacaaacctggcggctttaaggccagcaccggcttcggcagcaacaccaagaacaagaagatctacgacggcggagcacacaccgaggatgaggtgcagagctaccccagcaagcacgactacgtg (SEQ ID NO: 109)modClaudin 18 (CLDN18)AVTACQSLGFVVSLIEIVGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWHSCMRESSGFTECRGYFTLLELPAMLQAVQALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWLSTANMYTGMGEMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVYYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGAHTEDEVQSYPSKHDYV(SEQ ID NO: 110) modLY6Kgctctgctggcactgctgctggtggtggctttgcctagagtgtggaccgacgccaatctgacagtgcggcagagagatcctgaggacagccagagaaccgacgacggcgataacagagtgtggtgccacgtgtgcgagcgcgagaataccttcgagtgtcagaaccccagacggtgcaagtggaccgagccttactgtgtgatcgccgccgtgaaaatcttcccacggttcttcatggtggtcaagcagtgcagcgctggctgtgccgctatggaaagacccaagcctgaggaaaagcggttcctgctcgaggaacccatgctgttcttctacctgaagtgctgcaaaatctgctactgcaacctggaaggccctcctatcaacagcagcgtcctgaaagaatatgccggcagcatgggcgagtcttgtggtggactgtggctggccattctgctgctgcttgcctctattgccgcctctctgagcctgagc (SEQ ID NO: 111) modLY6KALLALLLVVALPRVWTDANLTVRQRDPEDSQRTDDGDNRVWCHVCERENTFECQNPRRCKWTEPYCVIAAVKIFPRFFMVVKQCSAGCAAMERPKPEEKRFLLEEPMLFFYLKCCKICYCNLEGPPINSSVLKEYAGSMGESCGGLWLAILLLLASIAASLSLS (SEQ ID NO: 112)modBORIS in modTBXT_BORISgccgccaccgagatcagcgtgctgagcgagcagttcaccaagatcaaagaattgaagctgatgctcgagaaggggctgaagaaagaagagaaggacggcgtctgccgcgagaagaatcacagaagccctagcgagctggaagcccagagaacatctggcgccttccaggacagcatcctggaagaagaggtggaactggttctggcccctctggaagagagcaagaagtacatcctgacactgcagaccgtgcacttcacctctgaagccgtgcagctccaggacatgagcctgctgtctatccagcagcaagagggcgtgcaggttgtggttcagcaacctggacctggactgctctggctgcaagagggacctagacagtccctgcagcagtgtgtggccatcagcatccagcaagagctgtatagccctcaagagatggaagtgctgcagtttcacgccctcgaagagaacgtgatggtggccatcgaggacagcaagctggctgtgtctctggccgaaacaaccggcctgatcaagctggaagaggaacaagagaagaaccagctgctggccgagaaaacaaaaaagcaactgttcttcgtggaaaccatgagcggcgacgagagaagcgacgagatcgtgctgacagtgtccaacagcaacgtggaagaacaagaggaccagcctaccgcctgtcaggccgatgccgagaaagccaagtttaccaagaaccagagaaagaccaagggcgccaagggcaccttccactgcaacgtgtgcatgttcaccagcagccggatgagcagcttcaactgccacatgaagacccacaccagcgagaagccccatctgtgtcacctgtgcctgaaaaccttccggacagtgacactgctgtggaactatgtgaacacccacacaggcacccggccttacaagtgcaacgactgcaacatggccttcgtgaccagcggagaactcgtgcggcacagaagatacaagcacacccacgagaaacccttcaagtgcagcatgtgcaaatacgcatccatggaagcctccaagctgaagtgccacgtgcgctctcacacaggcgagcaccctttccagtgctgtcagtgtagctacgccagccgggacacctataagctgaagcggcacatgagaacccactctggcgaaaagccctacgagtgccacatctgccacaccagattcacccagagcggcaccatgaagattcacatcctgcagaaacacggcaagaacgtgcccaagtaccagtgtcctcactgcgccaccattatcgccagaaagtccgacctgcgggtgcacatgaggaatctgcacgcctattctgccgccgagctgaaatgcagatactgcagcgccgtgttccacaagagatacgccctgatccagcaccagaaaacccacaagaacgagaagcggtttaagtgcaagcactgcagctacgcctgcaagcaagagcgccacatgatcgcccacatccacacacacaccggggagaagccttttacctgcctgagctgcaacaagtgcttccggcagaaacagctgctcaacgcccacttcagaaagtaccacgacgccaacttcatccccaccgtgtacaagtgctccaagtgcggcaagggcttcagccggtggatcaatctgcaccggcacctggaaaagtgcgagtctggcgaagccaagtctgccgcctctggcaagggcagaagaacccggaagagaaagcagaccatcctgaaagaggccaccaagagccagaaagaagccgccaagcgctggaaagaggctgccaacggcgacgaagctgctgccgaagaagccagcacaacaaagggcgaacagttccccgaagagatgttccctgtggcctgcagagaaaccacagccagagtgaagcaagaggtcgaccagggcgtgacctgcgagatgctgctgaacaccatggacaag (SEQ ID NO: 113)modFAPatgaagaccctggtcaagatcgtgtttggcgtggccacatctgccgtgctggctctgctggtcatgtgcattgtgctgcaccccagcagagtgcacaacagcgaagagaacaccatgcgggccctgacactgaaggacatcctgaacgtgaccttcagctacaagatattcttccccaactggatctccggccaagagtacctgcaccagagcgccgacaacaacatcgtgctgtacaacatcgagacaggccagagctacaccatcatgagcaaccggaccatgaagtccgtgaacgccagcaactacggactgagccccgattggcagttcgtgtacctggaaagcgactacagcaagctgtggcggtacagctacaccgccacctactacatctacgacctgagcaacggcgagttcgtgaagggcaacgagctgccccatcctatccagtacctgtgttggagccctgtgggctccaagctggcctacgtgtaccagaacaacatctacctgaagcagcggcctggcgaccctccattccagatcaccttcaacggcagagagaacaagatctttaacggcatccccgactgggtgtacgaggaagagatgctggccaccaaatacgccctgtggtggtcccctaacggcaagtttctggcctatgccgacttcaacgacacagacatccccgtgatcgcctacagctactacggcaatgagcagtaccccaggaccatcaacatcagctaccccaaagccggcgctaagaaccctgtcgtgcggatcttcatcatcgacaccacctatcctgtgtacgtgggccctcaagaggtgccagtgcctgccatgattgccagcagcgactactacttcagctggctgacctgggtcaccgacgagcgagtttgtctgcagtggctgaagcgggtgcagaacatcagcgtgctgagcatctgcgacttcagaaaggactggcagacatgggactgccccaacacacagcagcacatcgaggaaagcagaaccggctgggctggcggcttctttgtgtctacccctgtgttcagctacgacgccatcctgtactataagatcttcagcgacaaggacggctacaagcacatccactacatcaagtacaccgtcgagaacgtgatccagattaccagcggcaagtgggaagccatcaatatcttcagagtgatccagtacagcctgttctacagcagcaacgagttcgaggaataccccggcagacggaacatctacagaatcagcatcggcagctacccgcctagcaagaaatgcgtgacctgccacctgagaaaagagcggtgccagtactacacagccagcttctccaactacgccaagtactacgccctcgtgtgttacggccctggcatccctatcagcacactgcacgatggcagaaccgaccaagagatcaagatcctggaagaaaacaaagagctggaaaacgccctgaagaacatccagctgcctaaagaggaaatcaagaagctggaagtcgacgagatcaccctgtggtacaagatgatcctgcctcctcagttcgaccggtccaagaagtaccctctgctgatccaggtgtacggcggaccttgttctcagtctgtgcgctccgtgttcgccgtgaattggatcagctatctggccagcaaagaaggcatggttatcgccctggtggacggcagaggcacagcttttcaaggcgacaagctgctgtacgccgtgtatcagaaactgggcgtgtacgaagtggaagatcagatcaccgccgtgcggaagttcatcgagatgggcttcatcgacgagaagcggatcgccatctggggctggtcttacggcggctatattagctctctggccctggcctctggcaccggcctgtttaagtgtggaattgccgtggctcccgtgtccagctgggagtactataccagcgtgtacaccgagcggttcatgggcctgcctaccaaggacgacaacctggaacactacaagaactctaccgtgatggccagagccgagtacttccggaacgtggactacctgctgattcacggcaccgccgacgacaacgtgcacttccaaaacagcgcccagatcgctaaggccctcgtgaatgcccaggtggactttcaggccatgtggtacagcgaccagaaccacggactgtctggcctgagcaccaaccacctgtacacccacatgacccactttctgaaacagtgcttcagcctgagcgac (SEQ ID NO: 114)modFAPMKTLVKIVFGVATSAVLALLVMCIVLHPSRVHNSEENTMRALTLKDILNVTFSYKIFFPNWISGQEYLHQSADNNIVLYNIETGQSYTIMSNRTMKSVNASNYGLSPDWQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVKGNELPHPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYADFNDTDIPVIAYSYYGNEQYPRTINISYPKAGAKNPVVRIFIIDTTYPVYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNISVLSICDFRKDWQTWDCPNTQQHIEESRTGWAGGFFVSTPVFSYDAILYYKIFSDKDGYKHIHYIKYTVENVIQITSGKWEAINIFRVIQYSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSNYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYQKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYISSLALASGTGLFKCGIAVAPVSSWEYYTSVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD (SEQ ID NO: 115)modClaudin 18 in modFAP_Claudin 18gccgtcacagcctgtcagagcctgggctttgtggtgtccctgatcgagatcgtgggcatcattgccgctacctgcatggaccagtggtctacccaggacctgtataacaaccccgtgaccgccgtgttcaactaccaaggcctgtggcacagctgcatgagagagagcagcggcttcaccgagtgcaggggctactttaccctgctggaactgccagccatgctgcaggctgtgcaggcccttatgatcgtgggaattgtgctgggcgccatcggcctgctggtgtctatttttgccctgaagtgcatccggatcggcagcatggaagatagcgccaaggccaacatgaccctgacctccggcatcatgttcatcgtgtccggcctgtgtgccattgcaggcgtgtccgtgtttgccaatatgctcgtgaccaacttctggctgtccaccgccaacatgtacaccggcatgggcgagatggtgcagaccgtgcagacacggtacacatttggcgccgctctgtttgtcggatgggttgcaggcggactgactctgattggcggcgtgatgatgtgtatcgcctgcagaggactggcccctgaggaaacaaactacaaggccgtgtactaccacgccagcggacacagcgtggcatacaaaccaggcggctttaaggccagcacaggcttcggcagcaacaccaagaacaagaagatctacgacggcggagcccataccgaggatgaggtgcagagctaccctagcaagcacgactacgtg (SEQ ID NO: 116)

In some embodiments, provided herein is a vaccine composition comprisinga therapeutically effective amount of cells from at least two cancercell lines, wherein each cell line or a combination of the cell linesexpresses at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the TAAs ofTables 7-23. In other embodiments, the TAAs in Tables 7-23 are modifiedto include one or more NSMs as described herein. In some embodiments, atleast one cell line is modified to increase production of at least 1, 2,or 3 immunostimulatory factors, e.g., immunostimulatory factors fromTable 4. In some embodiments, a vaccine composition is providedcomprising a therapeutically effective amount of the cells from at leastone cancer cell line, wherein each cell line or combination of celllines is modified to reduce at least 1, 2, or 3 immunosuppressivefactors, e.g., immunosuppressive factors from Table 6. In someembodiments, a vaccine composition is provided comprising two cocktails,wherein each cocktail comprises three cell lines modified to express 1,2, or 3 immunostimulatory factors and to inhibit or reduce expression of1, 2, or 3 immunosuppressive factors, and wherein each cell lineexpresses at least 10 TAAs or TAAs comprising one or more NSMs.

Methods and assays for determining the presence or expression level of aTAA in a cell line according to the disclosure or in a tumor from asubject are known in the art. By way of example, Warburg-Christianmethod, Lowry Assay, Bradford Assay, spectrometry methods such as highperformance liquid chromatography (HPLC), liquid chromatography-massspectrometry (LC/MS), immunoblotting and antibody-based techniques suchas western blot, ELISA, immunoelectrophoresis, proteinimmunoprecipitation, flow cytometry, and protein immunostaining are allcontemplated by the present disclosure.

The antigen repertoire displayed by a patient's tumor can be evaluatedin some embodiments in a biopsy specimen using next generationsequencing and antibody-based approaches. Similarly, in someembodiments, the antigen repertoire of potential metastatic lesions canbe evaluated using the same techniques to determine antigens expressedby circulating tumor cells (CTCs). Assessment of antigen expression intumor biopsies and CTCs can be representative of a subset of antigensexpressed. In some embodiments, a subset of the antigens expressed by apatient's primary tumor and/or CTCs are identified and, as describedherein, informs the selection of cell lines to be included in thevaccine composition in order to provide the best possible match to theantigens expressed in a patient's tumor and/or metastatic lesions.

Embodiments of the present disclosure provides compositions of celllines that (i) are modified as described herein and (ii) express asufficient number and amount of TAAs such that, when administered to apatient afflicted with a cancer, cancers, or cancerous tumor(s), aTAA-specific immune response is generated.

Methods of Stimulating an Immune Response and Methods of Treatment

The vaccine compositions described herein may be administered to asubject in need thereof. Provided herein are methods for inducing animmune response in a subject, which involve administering to a subjectan immunologically effective amount of the genetically modified cells.Also provided are methods for preventing or treating a tumor in asubject by administering an anti-tumor effective amount of the vaccinecompositions described herein. Such compositions and methods may beeffective to prolong the survival of the subject.

According to various embodiments, administration of any one of thevaccine compositions provided herein can increase pro-inflammatorycytokine production (e.g., IFNγ secretion) by leukocytes. In someembodiments, administration of any one of the vaccine compositionsprovided herein can increase pro-inflammatory cytokine production (e.g.,IFNγ secretion) by leukocytes by at least 1.5-fold, 1.6-fold, 1.75-fold,2-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold ormore. In other embodiments, the IFNγ production is increased byapproximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or 25-fold or higher compared to unmodifiedcancer cell lines. Without being bound to any theory or mechanism, theincrease in pro-inflammatory cytokine production (e.g., IFNγ secretion)by leukocytes is a result of either indirect or direct interaction withthe vaccine composition.

In some embodiments, administration of any one of the vaccinecompositions provided herein comprising one or more modified cell linesas described herein can increase the uptake of cells of the vaccinecomposition by phagocytic cells, e.g., by at least 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold or more, as compared to acomposition that does not comprise modified cells.

In some embodiments, the vaccine composition is provided to a subject byan intradermal injection. Without being bound to any theory ormechanism, the intradermal injection, in at least some embodiments,generates a localized inflammatory response recruiting immune cells tothe injection site. Following administration of the vaccine, antigenpresenting cells (APCs) in the skin, such as Langerhans cells (LCs) anddermal dendritic cells (DCs), uptake the vaccine cell line components byphagocytosis and then migrate through the dermis to the draining lymphnode. At the draining lymph node, DCs or LCs that have phagocytized thevaccine cell line components are expected to prime naïve T cells and Bcells. Priming of naïve T and B cells is expected to initiate anadaptive immune response to tumor associated antigens (TAAs) expressedby the vaccine cell line components. Certain TAAs expressed by thevaccine cell line components are also expressed by the patient's tumor.Expansion of antigen specific T cells at the draining lymph node andtrafficking of these T cells to the tumor microenvironment (TME) isexpected to generate a vaccine-induced anti-tumor response.

According to various embodiments, immunogenicity of the allogenicvaccine composition can be further enhanced through geneticmodifications that reduce expression of immunosuppressive factors whileincreasing the expression or secretion of immunostimulatory signals.Modulation of these factors aims to enhance the uptake vaccine cell linecomponents by LCs and DCs in the dermis, trafficking of DCs and LCs tothe draining lymph node, T cell and B cell priming in the draining lymphnode, and, thereby resulting in more potent anti-tumor responses.

In some embodiments, the breadth of TAAs targeted in the vaccinecomposition can be increased through the inclusion of multiple celllines. For example, different histological subsets within a certaintumor type tend to express different TAA subsets. As a further example,in NSCLC, adenocarcinomas, and squamous cell carcinomas expressdifferent antigens. The magnitude and breadth of the adaptive immuneresponse induced by the vaccine composition can, according to someembodiments of the disclosure, be enhanced through the inclusion ofadditional cell lines expressing the same or different immunostimulatoryfactors. For example, expression of an immunostimulatory factor, such asIL-12, by one cell line within a cocktail of three cell lines can actlocally to enhance the immune responses to all cell lines delivered intothe same site. The expression of an immunostimulatory factor by morethan one cell line within a cocktail, such as GM-CSF, can increase theamount of the immunostimulatory factor in the injection site, therebyenhancing the immune responses induced to all components of thecocktail. The degree of HLA mismatch present within a vaccine cocktailmay further enhance the immune responses induced by that cocktail.

As described herein, in various embodiments, a method of stimulating animmune response specific to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more TAAs in a subject isprovided comprising administering a therapeutically effective amount ofa vaccine composition comprising modified cancer cell lines.

An “immune response” is a response of a cell of the immune system, suchas a B cell, T cell, or monocyte, to a stimulus, such as a cell orantigen (e.g., formulated as an antigenic composition or a vaccine). Animmune response can be a B cell response, which results in theproduction of specific antibodies, such as antigen specific neutralizingantibodies. An immune response can also be a T cell response, such as aCD4+ response or a CD8+ response. B cell and T cell responses areaspects of a “cellular” immune response. An immune response can also bea “humoral” immune response, which is mediated by antibodies. In somecases, the response is specific for a particular antigen (that is, an“antigen specific response”), such as one or more TAAs, and thisspecificity can include the production of antigen specific antibodiesand/or production of a cytokine such as interferon gamma which is a keycytokine involved in the generation of a Th₁ T cell response andmeasurable by ELISpot and flow cytometry.

Vaccine efficacy can be tested by measuring the T cell response CD4+ andCD8+ after immunization, using flow cytometry (FACS) analysis, ELISpotassay, or other method known in the art. Exposure of a subject to animmunogenic stimulus, such as a cell or antigen (e.g., formulated as anantigenic composition or vaccine), elicits a primary immune responsespecific for the stimulus, that is, the exposure “primes” the immuneresponse. A subsequent exposure, e.g., by immunization, to the stimuluscan increase or “boost” the magnitude (or duration, or both) of thespecific immune response. Thus, “boosting” a preexisting immune responseby administering an antigenic composition increases the magnitude of anantigen (or cell) specific response, (e.g., by increasing antibody titerand/or affinity, by increasing the frequency of antigen specific B or Tcells, by inducing maturation effector function, or a combinationthereof).

The immune responses that are monitored/assayed or stimulated by themethods described herein include, but not limited to: (a) antigenspecific or vaccine specific IgG antibodies; (b) changes in serumcytokine levels that may include and is not limited to: IL-1β, IL-4,IL-5, IL-6, IL-8, IL-10, IL-12, IL-17A, IL-20, IL-22, TNFα, IFNγ, TGFβ,CCLS, CXCL10; (c) IFNγ responses determined by ELISpot for CD4 and CD8 Tcell vaccine and antigen specific responses; (d) changes in IFNγresponses to TAA or vaccine cell components; (e) increased T cellproduction of intracellular cytokines in response to antigenstimulation: IFNγ, TNFα, and IL-2 and indicators of cytolytic potential:Granzyme A, Granzyme B, Perforin, and CD107a; (f) decreased levels ofregulatory T cells (Tregs), mononuclear monocyte derived suppressorcells (M-MDSCs), and polymorphonuclear derived suppressor cells(PMN-MDSCs); (g) decreased levels of circulating tumor cells (CTCs); (h)neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio(PLR); (i) changes in immune infiltrate in the TME; and (j) dendriticcell maturation.

Assays for determining the immune responses are described herein andwell known in the art. DC maturation can be assessed, for example, byassaying for the presence of DC maturation markers such as CD80, CD83,CD86, and MHC II. (See Dudek, A., et al., Front. Immunol., 4:438(2013)). Antigen specific or vaccine specific IgG antibodies can beassessed by ELISA or flow cytometry. Serum cytokine levels can bemeasured using a multiplex approach such as Luminex or Meso ScaleDiscovery Electrochemiluminescence (MSD). T cell activation and changesin lymphocyte populations can be measured by flow cytometry. CTCs can bemeasured in PBMCs using a RT-PCR based approach. The NLR and PLR ratioscan be determined using standard complete blood count (CBC) chemistrypanels. Changes in immune infiltrate in the TME can be assessed by flowcytometry, tumor biopsy and next-generation sequencing (NGS), orpositron emission tomography (PET) scan of a subject.

Given the overlap in TAA expression between cancers and tumors ofdifferent types, the present disclosure provides, in certainembodiments, compositions that can treat multiple different cancers. Forexample, one vaccine composition comprising two cocktails of three celllines each may be administered to a subject suffering from two or moretypes of cancers and said vaccine composition is effective at treatingboth, additional or all types of cancers. In exemplary embodiments, andin consideration of the TAA expression profile, the same vaccinecomposition comprising modified cancer cell lines is used to treatprostate cancer and testicular cancer, gastric and esophageal cancer, orendometrial, ovarian, and breast cancer in the same patient (ordifferent patients). TAA overlap can also occur within subsets of hottumors or cold tumors. For example, TAA overlap occurs in GBM and SCLC,both considered cold tumors. Exemplary TAAs included in embodiments ofthe vaccine composition include GP100, MAGE-A1, MAGE-A4, MAGE-A10,Sart-1, Sart-3, Trp-1, and Sox2. In some embodiments, cell linesincluded in the vaccine composition can be selected from two tumor typesof similar immune landscape to treat one or both of the tumor types inthe same individual.

As used herein, changes in or “increased production” of, for example acytokine such as IFNγ, refers to a change or increase above a control orbaseline level of production/secretion/expression and that is indicativeof an immunostimulatory response to an antigen or vaccine component.

Combination Treatments and Regimens

Formulations, Adjuvants, and Additional Therapeutic Agents

The compositions described herein may be formulated as pharmaceuticalcompositions. The term “pharmaceutically acceptable” as used hereinrefers to a pharmaceutically acceptable material, composition, orvehicle, such as a liquid or solid filler, diluent, excipient, solvent,or encapsulating material. Each component must be “pharmaceuticallyacceptable” in the sense of being compatible with the other ingredientsof a pharmaceutical formulation. It must also be suitable for use incontact with tissue, organs or other human component without excessivetoxicity, irritation, allergic response, immunogenicity, or otherproblems or complications, commensurate with a reasonable benefit/riskratio. (See Remington: The Science and Practice of Pharmacy, 21stEdition; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005;Handbook of Pharmaceutical Excipients, 5th Edition; Rowe et al., Eds.,The Pharmaceutical Press and the American Pharmaceutical Association:2005; and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and AshEds., Gower Publishing Company: 2007; Pharmaceutical Preformulation andFormulation, Gibson Ed., CRC Press LLC: Boca Raton, Fla., 2004)).

Embodiments of the pharmaceutical composition of the disclosure isformulated to be compatible with its intended route of administration(i.e., parenteral, intravenous, intra-arterial, intradermal,subcutaneous, oral, inhalation, transdermal, topical, intratumoral,transmucosal, intraperitoneal or intra-pleural, and/or rectaladministration). Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; dimethyl sulfoxide (DMSO); antibacterial agents suchas benzyl alcohol or methyl parabens; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid (EDTA); buffers such as acetates, citrates or phosphates, andagents for the adjustment of tonicity such as sodium chloride ordextrose. The pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide. The parenteral preparation can beenclosed in ampoules, disposable syringes, or one or more vialscomprising glass or polymer (e.g., polypropylene). The term “vial” asused herein means any kind of vessel, container, tube, bottle, or thelike that is adapted to store embodiments of the vaccine composition asdescribed herein.

In some embodiments, the composition further comprises apharmaceutically acceptable carrier. The term “carrier” as used hereinencompasses diluents, excipients, adjuvants, and combinations thereof.Pharmaceutically acceptable carriers are well known in the art (SeeRemington: The Science and Practice of Pharmacy, 21st Edition).Exemplary “diluents” include sterile liquids such as sterile water,saline solutions, and buffers (e.g., phosphate, tris, borate, succinate,or histidine). Exemplary “excipients” are inert substances that mayenhance vaccine stability and include but are not limited to polymers(e.g., polyethylene glycol), carbohydrates (e.g., starch, glucose,lactose, sucrose, or cellulose), and alcohols (e.g., glycerol, sorbitol,or xylitol).

In various embodiments, the vaccine compositions and cell linecomponents thereof are sterile and fluid to the extent that thecompositions and/or cell line components can be loaded into one or moresyringes. In various embodiments, the compositions are stable under theconditions of manufacture and storage and preserved against thecontaminating action of microorganisms such as bacteria and fungi. Insome embodiments, the carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion, by the use ofsurfactants, and by other means known to one of skill in the art.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In someembodiments, it may be desirable to include isotonic agents, forexample, sugars, polyalcohols such as manitol, sorbitol, and/or sodiumchloride in the composition. In some embodiments, prolonged absorptionof the injectable compositions can be brought about by including in thecomposition an agent that delays absorption, for example, aluminummonostearate and gelatin.

In some embodiments, sterile injectable solutions can be prepared byincorporating the active compound(s) in the required amount(s) in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. In certainembodiments, dispersions are prepared by incorporating the activecompound into a sterile vehicle that contains a basic dispersion mediumand the required other ingredients from those enumerated herein. In thecase of sterile powders for the preparation of sterile injectablesolutions, embodiments of methods of preparation include vacuum dryingand freeze-drying that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The innate immune system comprises cells that provide defense in anon-specific manner to infection by other organisms. Innate immunity ina subject is an immediate defense, but it is not long-lasting orprotective against future challenges. Immune system cells that generallyhave a role in innate immunity are phagocytic, such as macrophages anddendritic cells. The innate immune system interacts with the adaptive(also called acquired) immune system in a variety of ways.

In some embodiments, the vaccine compositions alone activate an immuneresponse (i.e., an innate immune response, an adaptive immune response,and/or other immune response). In some embodiments, one or moreadjuvants are optionally included in the vaccine composition or areadministered concurrently or strategically in relation to the vaccinecomposition, to provide an agent(s) that supports activation of innateimmunity in order to enhance the effectiveness of the vaccinecomposition. An “adjuvant” as used herein is an “agent” or substanceincorporated into the vaccine composition or administered simultaneouslyor at a selected time point or manner relative to the administration ofthe vaccine composition. In some embodiments, the adjuvant is a smallmolecule, chemical composition, or therapeutic protein such as acytokine or checkpoint inhibitor. A variety of mechanisms have beenproposed to explain how different agents function (e.g., antigen depots,activators of dendritic cells, macrophages). An agent may act to enhancean acquired immune response in various ways and many types of agents canactivate innate immunity. Organisms, like bacteria and viruses, canactivate innate immunity, as can components of organisms, chemicals suchas 2′-5′ oligo A, bacterial endotoxins, RNA duplexes, single strandedRNA and other compositions. Many of the agents act through a family ofmolecules referred to herein as “toll-like receptors” (TLRs). Engaging aTLR can also lead to production of cytokines and chemokines andactivation and maturation of dendritic cells, components involved indevelopment of acquired immunity. The TLR family can respond to avariety of agents, including lipoprotein, peptidoglycan, flagellin,imidazoquinolines, CpG DNA, lipopolysaccharide and double stranded RNA.These types of agents are sometimes called pathogen (ormicrobe)-associated molecular patterns. In some embodiments, theadjuvant is a TLR4 agonist.

One adjuvant that in some embodiments may be used in the vaccinecompositions is a monoacid lipid A (MALA) type molecule. An exemplaryMALA is MPL® adjuvant as described in, e.g., Ulrich J. T. and Myers, K.R., Chapter 21 in Vaccine Design, the Subunit and Adjuvant Approach,Powell, M. F. and Newman, M. J., eds. Plenum Press, NY (1995).

In other embodiments, the adjuvant may be “alum”, where this term refersto aluminum salts, such as aluminum phosphate and aluminum hydroxide.

In some embodiments, the adjuvant may be an emulsion having vaccineadjuvant properties. Such emulsions include oil-in-water emulsions.Incomplete Freund's adjuvant (IFA) is one such adjuvant. Anothersuitable oil-in-water emulsion is MF-59™ adjuvant which containssqualene, polyoxyethylene sorbitan monooleate (also known as Tween® 80surfactant) and sorbitan trioleate. Other suitable emulsion adjuvantsare Montanide™ adjuvants (Seppic Inc., Fairfield N.J.) includingMontanide™ ISA 50V which is a mineral oil-based adjuvant, Montanide™ ISA206, and Montanide™ IMS 1312. While mineral oil may be present in theadjuvant, in one embodiment, the oil component(s) of the compositions ofthe present disclosure are all metabolizable oils.

In some embodiments, the adjuvant may be AS02™ adjuvant or AS04™adjuvant. AS02™ adjuvant is an oil-in-water emulsion that contains bothMPL™ adjuvant and QS-21™ adjuvant (a saponin adjuvant discussedelsewhere herein). AS04™ adjuvant contains MPL™ adjuvant and alum. Theadjuvant may be Matrix-M™ adjuvant. The adjuvant may be a saponin suchas those derived from the bark of the Quillaja saponaria tree species,or a modified saponin, see, e.g., U.S. Pat. Nos. 5,057,540; 5,273,965;5,352,449; 5,443,829; and 5,560,398. The product QS-21™ adjuvant sold byAntigenics, Inc. (Lexington, Mass.) is an exemplary saponin-containingco-adjuvant that may be used with embodiments of the compositiondescribed herein. In other embodiments, the adjuvant may be one or acombination of agents from the ISCOM™ family of adjuvants, originallydeveloped by Iscotec (Sweden) and typically formed from saponins derivedfrom Quillaja saponaria or synthetic analogs, cholesterol, andphospholipid, all formed into a honeycomb-like structure.

In some embodiments, the adjuvant or agent may be a cytokine thatfunctions as an adjuvant, see, e.g., Lin R. et al. Clin. Infec. Dis.21(6):1439-1449 (1995); Taylor, C. E., Infect. Immun. 63(9):3241-3244(1995); and Egilmez, N. K., Chap. 14 in Vaccine Adjuvants and DeliverySystems, John Wiley & Sons, Inc. (2007). In various embodiments, thecytokine may be, e.g., granulocyte-macrophage colony-stimulating factor(GM-CSF); see, e.g., Change D. Z. et al. Hematology 9(3):207-215 (2004),Dranoff, G. Immunol. Rev. 188:147-154 (2002), and U.S. Pat. No.5,679,356; or an interferon, such as a type I interferon, e.g.,interferon-α (IFN-α) or interferon-β (IFN-β), or a type II interferon,e.g., interferon-γ (IFNγ), see, e.g., Boehm, U. et al. Ann. Rev.Immunol. 15:749-795 (1997); and Theofilopoulos, A. N. et al. Ann. Rev.Immunol. 23:307-336 (2005); an interleukin, specifically includinginterleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-2 (IL-2);see, e.g., Nelson, B. H., J. Immunol. 172(7): 3983-3988 (2004);interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-12 (IL-12); see,e.g., Portielje, J. E., et al., Cancer Immunol. Immunother. 52(3):133-144 (2003) and Trinchieri. G. Nat. Rev. Immunol. 3(2):133-146(2003); interleukin-15 (11-15), interleukin-18 (IL-18); fetal livertyrosine kinase 3 ligand (Flt3L), or tumor necrosis factor α (TNFα).

In some embodiments, the adjuvant may be unmethylated CpG dinucleotides,optionally conjugated to the antigens described herein.

Examples of immunopotentiators that may be used in the practice of thecompositions and methods described herein as adjuvants include: MPL™;MDP and derivatives; oligonucleotides; double-stranded RNA; alternativepathogen-associated molecular patterns (PAMPS); saponins; small-moleculeimmune potentiators (SMIPs); cytokines; and chemokines.

When two or more adjuvants or agents are utilized in combination, therelative amounts of the multiple adjuvants may be selected to achievethe desired performance properties for the composition which containsthe adjuvants, relative to the antigen alone. For example, an adjuvantcombination may be selected to enhance the antibody response of theantigen, and/or to enhance the subject's innate immune system response.Activating the innate immune system results in the production ofchemokines and cytokines, which in turn may activate an adaptive(acquired) immune response. An important consequence of activating theadaptive immune response is the formation of memory immune cells so thatwhen the host re-encounters the antigen, the immune response occursquicker and generally with better quality. In some embodiments, theadjuvant(s) may be pre-formulated prior to their combination with thecompositions described herein.

Embodiments of the vaccine compositions described herein may beadministered simultaneously with, prior to, or after administration ofone or more other adjuvants or agents, including therapeutic agents. Incertain embodiments, such agents may be accepted in the art as astandard treatment or prevention for a particular cancer. Exemplaryagents contemplated include cytokines, growth factors, steroids, NSAIDs,DMARDs, anti-inflammatories, immune checkpoint inhibitors,chemotherapeutics, radiotherapeutic, or other active and ancillaryagents. In other embodiments, the agent is one or more isolated TAA asdescribed herein.

In some embodiments, a vaccine composition provided herein isadministered to a subject that has not previously received certaintreatment or treatments for cancer or other disease or disorder. As usedherein, the phrase “wherein the subject refrains from treatment withother vaccines or therapeutic agents” refers to a subject that has notreceived a cancer treatment or other treatment or procedure prior toreceiving a vaccine of the present disclosure. In some embodiments, thesubject refrains from receiving one or more therapeutic vaccines (e.g.flu vaccine, covid-19 vaccine such as AZD1222, BNT162b2, mRNA-1273, andthe like) prior to the administration of the therapeutic vaccine asdescribed in various embodiments herein. In some embodiments, thesubject refrains from receiving one or more antibiotics prior to theadministration of the therapeutic vaccine as described in variousembodiments herein. “Immune tolerance” is a state of unresponsiveness ofthe immune system to substances, antigens, or tissues that have thepotential to induce an immune response. The vaccine compositions of thepresent disclosure, in certain embodiments, are administered to avoidthe induction of immune tolerance or to reverse immune tolerance.

In various embodiments, the vaccine composition is administered incombination with one or more active agents used in the treatment ofcancer, including one or more chemotherapeutic agents. Examples of suchactive agents include alkylating agents such as thiotepa andcyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.,paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) andpaclitaxel protein-bound particles (ABRAXANE®) and doxetaxel (TAXOTERE®,Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine;6-thioguanine; mercaptopurine; methotrexate; platinum analogs such ascisplatin and carboplatin; vinblastine, docetaxel, platinum; etoposide(VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylomithine (DMFO); retinoic acid derivatives such asTARGRETIN™ (bexarotene), PANRETIN™ (alitretinoin); and ONTAK (denileukindiftitox); esperamicins; capecitabine; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Also included in thisdefinition are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Further cancer activeagents include sorafenib and other protein kinase inhibitors such asafatinib, axitinib, bevacizumab, cetuximab, crizotinib, dasatinib,erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib,mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab,ruxolitinib, trastuzumab, vandetanib, vemurafenib, and sunitinib;sirolimus (rapamycin), everolimus and other mTOR inhibitors.

In further embodiments, the vaccine composition is administered incombination with a TLR4 agonist, TLR8 agonist, or TLR9 agonist. Such anagonist may be selected from peptidoglycan, polyl:C, CpG, 3M003,flagellin, and Leishmania homolog of eukaryotic ribosomal elongation andinitiation factor 4a (LeIF).

In some embodiments, the vaccine composition is administered incombination with a cytokine as described herein. In some embodiments,the compositions disclosed herein may be administered in conjunctionwith molecules targeting one or more of the following: Adhesion:MAdCAM1, ICAM1, VCAM1, CD103; Inhibitory Mediators: IDO, TDO;MDSCs/Tregs: NOS1, arginase, CSFR1, FOXP3, cyclophosphamide, PI3Kgamma,PI3Kdelta, tasquinimod; Immunosuppression: TGFβ, IL-10; Priming andPresenting: BATF3, XCR1/XCL1, STING, INFalpha; Apoptotic Recycling:IL-6, surviving, IAP, mTOR, MCL1, PI3K; T-Cell Trafficking: CXCL9/10/11,CXCL1/13, CCL2/5, anti-LIGHT, anti-CCR5; Oncogenic Activation:WNT-beta-cat, MEK, PPARgamma, FGFR3, TKIs, MET; EpigeneticReprogramming: HDAC, HMA, BET; Angiogenesis immune modulation:VEGF(alpha, beta, gamma); Hypoxia: HIF1alpha, adenosine, anitADORA2A,anti-CD73, and anti-CD39.

In certain embodiments, the compositions disclosed herein may beadministered in conjunction with a histone deacetylase (HDAC) inhibitor.HDAC inhibitors include hydroxamates, cyclic peptides, aliphatic acidsand benzamides. Illustrative HDAC inhibitors contemplated for use hereininclude, but are not limited to, Suberoylanilide hydroxamic acid(SAHANorinostat/Zolinza), Trichostatin A (TSA), PXD-101, Depsipeptide(FK228/romidepsin/ISTODAX®), panobinostat (LBH589), MS-275, Mocetinostat(MGCD0103), ACY-738, TMP195, Tucidinostat, valproic acid, sodiumphenylbutyrate, 5-aza-2′-deoxycytidine (decitabine). See e.g., Kim andBae, Am J Transl Res 2011; 3(2):166-179; Odunsi et al., Cancer ImmunolRes. 2014 Jan. 1; 2(1): 37-49. Other HDAC inhibitors include Vorinostat(SAHA, M K0683), Entinostat (MS-275), Panobinostat (LBH589),Trichostatin A (TSA), Mocetinostat (MGCD0103), ACY-738, Tucidinostat(Chidamide), TMP195, Citarinostat (ACY-241), Belinostat (PXD101),Romidepsin (FK228, Depsipeptide), MC1568, Tubastatin A HCl, Givinostat(ITF2357), Dacinostat (LAQ824), CUDC-101, Quisinostat (JNJ-26481585)2HCl, Pracinostat (SB939), PCI-34051, Droxinostat, Abexinostat(PCI-24781), RGFP966, AR-42, Ricolinostat (ACY-1215), Valproic acidsodium salt (Sodium valproate), Tacedinaline (CI994), CUDC-907, Sodiumbutyrate, Curcumin, M344, Tubacin, RG2833 (RGFP109), Resminostat,Divalproex Sodium, Scriptaid, and Tubastatin A.

In certain embodiments, the vaccine composition is administered incombination with chloroquine, a lysosomotropic agent that preventsendosomal acidification and which inhibits autophagy induced by tumorcells to survive accelerated cell growth and nutrient deprivation. Moregenerally, the compositions comprising heterozygous viral vectors asdescribed herein may be administered in combination with active agentsthat act as autophagy inhibitors, radiosensitizers or chemosensitizers,such as chloroquine, misonidazole, metronidazole, and hypoxiccytotoxins, such as tirapazamine. In this regard, such combinations of aheterozygous viral vector with chloroquine or other radio or chemosensitizer, or autophagy inhibitor, can be used in further combinationwith other cancer active agents or with radiation therapy or surgery.

In other embodiments, the vaccine composition is administered incombination with one or more small molecule drugs that are known toresult in killing of tumor cells with concomitant activation of immuneresponses, termed “immunogenic cell death”, such as cyclophosphamide,doxorubicin, oxaliplatin and mitoxantrone. Furthermore, combinationswith drugs known to enhance the immunogenicity of tumor cells such aspatupilone (epothilone B), epidermal-growth factor receptor(EGFR)-targeting monoclonal antibody 7A7.27, histone deacetylaseinhibitors (e.g., vorinostat, romidepsin, panobinostat, belinostat, andentinostat), the n3-polyunsaturated fatty acid docosahexaenoic acid,furthermore proteasome inhibitors (e.g., bortezomib), shikonin (themajor constituent of the root of Lithospermum erythrorhizon,) andoncolytic viruses, such as TVec (talimogene laherparepvec). In someembodiments, the compositions comprising heterozygous viral vectors asdescribed herein may be administered in combination with epigenetictherapies, such as DNA methyltransferase inhibitors (e.g., decitabine,5-aza-2′-deoxycytidine) which may be administered locally orsystemically.

In other embodiments, the vaccine composition is administered incombination with one or more antibodies that increase ADCC uptake oftumor by DCs. Thus, embodiments of the present disclosure contemplatecombining cancer vaccine compositions with any molecule that induces orenhances the ingestion of a tumor cell or its fragments by an antigenpresenting cell and subsequent presentation of tumor antigens to theimmune system. These molecules include agents that induce receptorbinding (e.g., Fc or mannose receptors) and transport into the antigenpresenting cell such as antibodies, antibody-like molecules,multi-specific multivalent molecules and polymers. Such molecules mayeither be administered intratumorally with the composition comprisingheterozygous viral vector or administered by a different route. Forexample, a composition comprising heterozygous viral vector as describedherein may be administered intratumorally in conjunction withintratumoral injection of rituximab, cetuximab, trastuzumab, Campath,panitumumab, ofatumumab, brentuximab, pertuzumab, Ado-trastuzumabemtansine, Obinutuzumab, anti-HER1, -HER2, or -HER3 antibodies (e.g.,MEHD7945A; MM-111; MM-151; MM-121; AMG888), anti-EGFR antibodies (e.g.,nimotuzumab, ABT-806), or other like antibodies. Any multivalentscaffold that is capable of engaging Fc receptors and other receptorsthat can induce internalization may be used in the combination therapiesdescribed herein (e.g., peptides and/or proteins capable of bindingtargets that are linked to Fc fragments or polymers capable of engagingreceptors).

In certain embodiments, the vaccine composition may be further combinedwith an inhibitor of ALK, PARP, VEGFRs, EGFR, FGFR1-3, HIF1α, PDGFR1-2,c-Met, c-KIT, Her2, Her3, AR, PR, RET, EPHB4, STAT3, Ras, HDAC1-11,mTOR, and/or CXCR4.

In certain embodiments, a cancer vaccine composition may be furthercombined with an antibody that promotes a co-stimulatory signal (e.g.,by blocking inhibitory pathways), such as anti-CTLA-4, or that activatesco-stimulatory pathways such as an anti-CD40, anti-CD28, anti-ICOS,anti-OX40, anti-CD27, anti-ICOS, anti-CD127, anti-GITR, IL-2, IL-7,IL-15, IL-21, GM-CSF, IL-12, and INFα.

Checkpoint Inhibitors

In certain embodiments, a checkpoint inhibitor molecule is administeredin combination with the vaccine compositions described herein. Immunecheckpoints refer to a variety of inhibitory pathways of the immunesystem that are crucial for maintaining self-tolerance and formodulating the duration and amplitude of an immune responses. Tumors usecertain immune-checkpoint pathways as a major mechanism of immuneresistance, particularly against T cells that are specific for tumorantigens. (See Pardoll, 2012 Nature 12:252; Chen and Mellman Immunity39:1 (2013)). Immune checkpoint inhibitors include any agent that blocksor inhibits in a statistically significant manner, the inhibitorypathways of the immune system. Such inhibitors may include antibodies,or antigen binding fragments thereof, that bind to and block or inhibitimmune checkpoint receptors or antibodies that bind to and block orinhibit immune checkpoint receptor ligands. Illustrative immunecheckpoint molecules that may be targeted for blocking or inhibitioninclude, but are not limited to, CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L),PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3,B7H4, VISTA, KIR, BTLA, SIGLEC9, 2B4 (belongs to the CD2 family ofmolecules and is expressed on all NK, γδ, and memory CD8+ (αβ) T cells),CD160 (also referred to as BY55), and CGEN-15049. Immune checkpointinhibitors include antibodies, or antigen binding fragments thereof, orother binding proteins, that bind to and block or inhibit the activityof one or more of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM,TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, BTLA, SIGLEC9, 2B4,CD160, and CGEN-15049.

Illustrative immune checkpoint inhibitors include anti-PD1, anti-PDL1,and anti-PDL2 agents such as A167, AB122, ABBV-181, ADG-104, AK-103,AK-105, AK-106, AGEN2034, AM0001, AMG-404, ANB-030, APL-502, APL-501,zimberelimab, atezolizumab, AVA-040, AVA-040-100, avelumab, balstilimab,BAT-1306, BCD-135, BGB-A333, BI-754091, budigalimab, camrelizumab,CB-201, CBT-502, CCX-4503, cemiplimab, cosibelimab, cetrelimab, CS-1001,CS-1003, CX-072, CX-188, dostarlimab, durvalumab, envafolimab,sugemalimab, HBM9167, F-520, FAZ-053, genolimzumab, GLS-010, GS-4224,hAB21, HLX-10, HLX-20, HS-636, HX-008, IMC-001, IMM-25, INCB-86550,JS-003, JTX-4014, JYO-34, KL-A167, LBL-006, lodapolimab, LP-002,LVGN-3616, LYN-00102, LMZ-009, MAX-10181, MEDI-0680, MGA-012(Retifanlimab), MSB-2311, nivolumab, pembrolizumab, prolgolimab,prololimab, sansalimab, SCT-110A, SG-001, SHR-1316, sintilimab,spartalizumab, RG6084, RG6139, RG6279, CA-170, CA-327, STI-3031,toleracyte, toca 521, Sym-021, TG-1501, tislelizumab, toripalimab,TT-01, ZKAB-001, and the anti-PD-1 antibodies capable of blockinginteraction with its ligands PD-L1 and PD-L2 described inWO/2017/124050.

Illustrative multi-specific immune checkpoint inhibitors, where at leastone target is anti-PD1, anti-PDL1, or anti-PDL2, include ABP-160(CD47×PD-L1), AK-104 (PD-1×CTLA-4), AK-112 (PD-1×VEGF), ALPN-202(PD-L1×CTLA-4×CD28), AP-201 (PD-L1×OX-40), AP-505 (PD-L1×VEGF), AVA-0017(PD-L1×LAG-3), AVA-0021 (PD-L1×LAG-3), AUPM-170 (PD-L1×VISTA), BCD-217(PD-1×CTLA-4), BH-2950 (PD-1×HER2), BH-2996h (PD-1×PD-L1), BH-29xx(PD-L1×CD47), bintrafusp alfa (PD-L1×TGFβ), CB-213 (PD-1×LAG-3), CDX-527(CD27×PD-L1), CS-4100 (PD-1×PD-L1), DB-001 (PD-L1×HER2), DB-002(PD-L1×CTLA-4), DSP-105 (PD-1×4-1BBL), DSP-106, (PD-1×CD70), FS-118(LAG-3×PD-L1), FS-222 (CD137/4-1BB×PD-L1), GEN-1046 (PD-L1×CD137/4-1BB),IBI-318 (PD-1×PD-L1), IBI-322 (PD-L1×CD-47), KD-033 (PD-L1×IL-15),KN-046 (PD-L1×CTLA-4), KY-1043 (PD-L1×IL-2), LY-3434172 (PD-1×PD-L1),MCLA-145 (PD-L1×CD137), MEDI-5752 (PD-1×CTLA-4), MGD-013 (PD-1×LAG-3),MGD-019 (PD-1×CTLA-4), ND-021 (PD-L1×4-1BB×HSA), OSE-279 (PD-1×PD-L1),PRS-332 (PD-1×HER2), PRS-344 (PD-L1×CD137), PSB-205 (PD-1×CTLA-4),R-7015 (PD-L1×TGFβ), RO-7121661 (PD-1×TIM-3), RO-7247669 (PD-1×LAG-3),SHR-1701 (PD-L1×TGFβ2), SL-279252 (PD-1×OX40L), TSR-075 (PD-1×LAG-3),XmAb-20717 (CTLA-4×PD-1), XmAb-23104 (PD-1×ICOS), and Y-111(PD-L1×CD-3).

Additional illustrative immune checkpoint inhibitors include anti-CTLA4agents such as: ADG-116, AGEN-2041, BA-3071, BCD-145, BJ-003,BMS-986218, BMS-986249, BPI-002, CBT-509, CG-0161, Olipass-1, HBM-4003,HLX-09, IBI-310, ipilimumab, JS-007, KN-044, MK-1308, ONC-392,REGN-4659, RP-2, tremelimumab, and zalifrelimab. Additional illustrativemulti-specific immune checkpoint inhibitors, where at least one targetis anti-CTLA4, include: AK-104 (PD-1×CTLA-4), ALPN-202(PD-L1×CTLA-4×CD28), ATOR-1015 (CTLA-4×OX40), ATOR-1144 (CTLA-4×GITR),BCD-217 (PD-1×CTLA-4), DB-002 (PD-L1×CTLA-4), FPT-155 (CD28×CTLA-4),KN-046 (PD-L1×CTLA-4),), MEDI-5752 (PD-1×CTLA-4), MGD-019 (PD-1×CTLA-4),PSB-205 (PD-1×CTLA-4), XmAb-20717 (CTLA-4×PD-1), and XmAb-22841(CTLA-4×LAG-3). Additional illustrative immune checkpoint inhibitorsinclude anti-LAG3 agents such as BI-754111, BJ-007, eftilagimod alfa,GSK-2831781, HLX-26, IBI-110, IMP-701, IMP-761, INCAGN-2385, LBL-007,MK-4280, REGN-3767, relatlimab, Sym-022, TJ-A3, and TSR-033. Additionalillustrative multi-specific immune checkpoint inhibitors, where at leastone target is anti-LAG3, include: CB-213 (PD-1×LAG-3), FS-118(LAG-3×PD-L1), MGD-013 (PD-1×LAG-3), AVA-0017 (PD-L1×LAG-3), AVA-0021(PD-L1×LAG- 3), RO-7247669 (PD-1×LAG-3), TSR-075 (PD-1×LAG-3), andXmAb-22841 (CTLA-4×LAG-3). Additional illustrative immune checkpointinhibitors include anti-TIGIT agents such as AB-154, ASP8374, BGB-A1217,BMS-986207, CASC-674, COM-902, EOS-884448, HLX-53, IBI-939, JS-006,MK-7684, NB-6253, RXI-804, tiragolumab, and YH-29143. Additionalillustrative multi-specific immune checkpoint inhibitors, where at leastone target is anti-TIGIT are contemplated. Additional illustrativeimmune checkpoint inhibitors include anti-TIM3 agents such as: BGB-A425,BMS-986258, ES-001, HLX-52, INCAGN-2390, LBL-003, LY-3321367, MBG-453,SHR-1702, Sym-023, and TSR-022. Additional illustrative multi-specificimmune checkpoint inhibitors, where at least one target is anti-TIM3,include: AUPM-327 (PD-L1×TIM-3), and RO-7121661 (PD-1×TIM-3). Additionalillustrative immune checkpoint inhibitors include anti-VISTA agents suchas: HMBD-002, and PMC-309. Additional illustrative multi-specific immunecheckpoint inhibitors, where at least one target is anti-VISTA, includeCA-170 (PD-L1×VISTA). Additional illustrative immune checkpointinhibitors include anti-BTLA agents such as: JS-004. Additionalillustrative multi-specific immune checkpoint inhibitors, where at leastone target is anti-BTLA are contemplated. Illustrative stimulatoryimmune checkpoints include anti-OX40 agents such as ABBV-368,GSK-3174998, HLX-51, IBI-101, INBRX-106, INCAGN-1949, INV-531, JNJ-6892,and KHK-4083. Additional illustrative multi-specific stimulatory immunecheckpoints, where at least one target is anti-OX40, include AP-201(PD-L1×OX-40), APVO-603 (CD138/4-1BB×OX-40), ATOR-1015 (CTLA-4×OX-40),and FS-120 (OX40×CD137/4-1BB). Additional illustrative stimulatoryimmune checkpoints include anti-GITR agents such as BMS-986256, CK-302,GWN-323, INCAGN-1876, MK-4166, PTZ-522, and TRX-518. Additionalillustrative multi-specific stimulatory immune checkpoints, where atleast one target is anti-GITR, include ATOR-1144 (CTLA-4×GITR).Additional illustrative stimulatory immune checkpoints includeanti-CD137/4-1BB agents such a: ADG-106, AGEN-2373, AP-116, ATOR-1017,BCY-3814, CTX-471, EU-101, LB-001, LVGN-6051, RTX-4-1BBL, SCB-333,urelumab, utomilumab, and WTiNT. Additional illustrative multi-specificstimulatory immune checkpoints, where at least one target isanti-CD137/4-1BB, include ALG.APV-527 (CD137/4-1BB×5T4), APVO-603(CD137/4-1BB×OX40), BT-7480 (Nectin-4×CD137/4-1BB), CB-307(CD137/4-1BB×PSMA), CUE-201 (CD80×CD137/4-1BB), DSP-105(PD-1×CD137/4-1BB), FS-120 (OX40×CD137/4-1BB), FS-222(PD-L1×CD137/4-1BB), GEN-1042 (CD40×CD137/4-1BB), GEN-1046(PD-L1×CD137/4-1BB), INBRX-105 (PD-L1×CD137/4-1BB), MCLA-145(PD-L1×CD137/4-1BB), MP-0310 (CD137/4-1BB×FAP), ND-021(PD-L1×CD137/4-1BB×HSA), PRS-343 (CD137/4-1BB×HER2), PRS-342(CD137/4-1BB×GPC3), PRS-344 (CD137/4-1BB×PD-L1), RG-7827 (FAP×4-1BBL),and RO-7227166 (CD-19×4-1BBL).

Additional illustrative stimulatory immune checkpoints include anti-ICOSagents such as BMS-986226, GSK-3359609, KY-1044, and vopratelimab.Additional illustrative multi-specific stimulatory immune checkpoints,where at least one target is anti-ICOS, include XmAb-23104 (PD-1×ICOS).Additional illustrative stimulatory immune checkpoints includeanti-CD127 agents such as MD-707 and OSE-703. Additional illustrativemulti-specific stimulatory immune checkpoints, where at least one targetis anti-CD127 are contemplated. Additional illustrative stimulatoryimmune checkpoints include anti-CD40 agents such as ABBV-428, ABBV-927,APG-1233, APX-005M, BI-655064, bleselumab, CD-40GEX, CDX-1140,LVGN-7408, MEDI-5083, mitazalimab, and selicrelumab. AdditionalIllustrative multi-specific stimulatory immune checkpoints, where atleast one target is anti-CD40, include GEN-1042 (CD40×CD137/4-1BB).Additional illustrative stimulatory immune checkpoints include anti-CD28agents such as FR-104 and theralizumab. Additional illustrativemulti-specific stimulatory immune checkpoints, where at least one targetis anti-CD28, include ALPN-101 (CD28×ICOS), ALPN-202 (PD-L1×CD28),CUE-201 (CD80×CD137/4-1BB), FPT-155 (CD28×CTLA-4), and REGN-5678(PSMA×CD28). Additional illustrative stimulatory immune checkpointsinclude anti-CD27 agents such as: HLX-59 and varlilumab. Additionalillustrative multi-specific stimulatory immune checkpoints, where atleast one target is anti-CD27, include DSP-160 (PD-L1×CD27/CD70) andCDX-256 (PD-L1×CD27). Additional illustrative stimulatory immunecheckpoints include anti-IL-2 agents such as ALKS-4230, BNT-151,CUE-103, NL-201, and THOR-707. Additional illustrative multi-specificstimulatory immune checkpoints, where at least one target is anti-IL-2,include CUE-102 (IL-2×WT1). Additional illustrative stimulatory immunecheckpoints include anti-IL-7 agents such as BNT-152. Additionalillustrative multi-specific stimulatory immune checkpoints, where atleast one target is anti-IL-7 are contemplated. Additional illustrativestimulatory immune checkpoints include anti-IL-12 agents such as AK-101,M-9241, and ustekinumab. Additional illustrative multi-specificstimulatory immune checkpoints, where at least one target is antilL-12are contemplated.

As described herein, the present disclosure provides methods ofadministering vaccine compositions, cyclophosphamide, checkpointinhibitors, and/or other therapeutic agents such as Treg inhibitors.Treg inhibitors are known in the art and include, for example,bempegaldesleukin, fludarabine, gemcitabine, mitoxantrone, CyclosporineA, tacrolimus, paclitaxel, imatinib, dasatinib, bevacizumab, idelalisib,anti-CD25, anti-folate receptor 4, anti-CTLA4, anti-GITR, anti-OX40,anti-CCR4, anti-CCR5, anti-CCR8, or TLR8 ligands.

Dosing

A “dose” or “unit dose” as used herein refers to one or more vaccinecompositions that comprise therapeutically effective amounts of one morecell lines. A dose can be a single vaccine composition, two separatevaccine compositions, or two separate vaccine compositions plus one ormore compositions comprising one or more therapeutic agents describedherein. When in separate compositions, the two or more compositions ofthe “dose” are meant to be administered “concurrently”. In someembodiments, the two or more compositions are administered at differentsites on the subject (e.g., arm, thigh, or back). As used herein,“concurrent” administration of two compositions or therapeutic agentsindicates that within about 30 minutes of administration of a firstcomposition or therapeutic agent, the second composition or therapeuticagent is administered. In cases where more than two compositions and/ortherapeutic agents are administered concurrently, each composition oragent is administered within 30 minutes, wherein timing of suchadministration begins with the administration of the first compositionor agent and ends with the beginning of administration of the lastcomposition or agent. In some cases, concurrent administration can becompleted (i.e., administration of the last composition or agent begins)within about 30 minutes, or within 15 minutes, or within 10 minutes, orwithin 5 minutes of start of administration of first composition oragent. Administration of a second (or multiple) therapeutic agents orcompositions “prior to” or “subsequent to” administration of a firstcomposition means that the administration of the first composition andanother therapeutic agent is separated by at least 30 minutes, e.g., atleast 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, atleast 8 hours, at least 10 hours, at least 12 hours, at least 18 hours,at least 24 hours, or at least 48 hours.

The amount (e.g., number) of cells from the various individual celllines in the vaccine compositions can be equal (as defined herein),approximately (as defined herein) equal, or different. In variousembodiments, each cell line of a vaccine composition is present in anapproximately equal amount. In other embodiments, 2 or 3 cell lines ofone vaccine composition are present in approximately equal amounts and 2or 3 different cell lines of a second composition are present inapproximately equal amounts.

In some embodiments, the number of cells from each cell line (in thecase where multiple cell lines are administered), is approximately5.0×10⁵, 1.0×10⁶, 2.0×10⁶, 3.0×10⁶, 4.0×10⁶, 5.0×10⁶, 6.0×10⁶, 7.0×10⁶,8×10⁶, 9.0×10⁶, 1.0×10⁷, 2.0×10⁷, 3.0×10⁷, 4.0×10⁷, 5.0×10⁷, 6.0×10⁷,8.0×10⁷, 9.0×10⁷, 1.0×10⁸, 2.0×10⁸, 3.0×10⁸, 4.0×10⁸ or 5.0×10⁸ cells.In one embodiment, approximately 10 million (e.g., 1.0×10⁷) cells fromone cell line are contemplated. In another embodiment, where 6 separatecell lines are administered, approximately 10 million cells from eachcell line, or 60 million (e.g., 6.0×10⁷) total cells are contemplated.

The total number of cells administered in a vaccine composition, e.g.,per administration site, can range from 1.0×10⁶ to 3.0×10⁸. For example,in some embodiments, 2.0×10⁶, 3.0×10⁶, 4.0×10⁶, 5.0×10⁶, 6.0×10⁶,7.0×10⁶, 8×10⁶, 9.0×10⁶, 1.0×10⁷, 2.0×10⁷, 3.0×10⁷, 4.0×10⁷, 5.0×10⁷,6.0×10⁷, 8.0×10⁷, 9.0×10⁷, 1.0×10⁸, 2.0×10⁸, or 3.0×10⁸ cells areadministered.

As described herein, the number of cell lines contained with eachadministration of a cocktail or vaccine composition can range from 1 to10 cell lines. In some embodiments, the number of cells from each cellline are not equal, and different ratios of cell lines are included inthe cocktail or vaccine composition. For example, if one cocktailcontains 5.0×10⁷ total cells from 3 different cell lines, there could be3.33×10⁷ cells of one cell line and 8.33×10⁶ of the remaining 2 celllines.

The vaccine compositions and compositions comprising additionaltherapeutic agents (e.g., chemotherapeutic agents, checkpointinhibitors, and the like) may be administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional,intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal,intracardial, intraarterial and sublingual injection or infusiontechniques. Also envisioned are embodiments where the vaccinecompositions and compositions comprising additional therapeutic agents(e.g., chemotherapeutic agents, checkpoint inhibitors, and the like) areadministered intranodally or intratumorally.

In some embodiments, the vaccine compositions are administeredintradermally. In related embodiments, the intradermal injectioninvolves injecting the cocktail or vaccine composition at an angle ofadministration of 5 to 15 degrees.

The injections (e.g., intradermal or subcutaneous injections), can beprovided at a single site (e.g. arm, thigh or back), or at multiplesites (e.g. arms and thighs). In some embodiments, the vaccinecomposition is administered concurrently at two sites, where each sitereceives a vaccine composition comprising a different composition (e.g.,cocktail). For example, in some embodiments, the subject receives acomposition comprising three cell lines in the arm, and three different,or partially overlapping cell lines in the thigh. In some embodiments,the subject receives a composition comprising one or more cell linesconcurrently in each arm and in each thigh.

In some embodiments, the subject receives multiple doses of the cocktailor vaccine composition and the doses are administered at different siteson the subject to avoid potential antigen competition at certain (e.g.,draining) lymph nodes. In some embodiments, the multiple doses areadministered by alternating administration sites (e.g. left arm andright arm, or left thigh and right thigh) on the subject between doses.In some embodiments, the multiple doses are administered as follows: afirst dose is administered in one arm, and second dose is administeredin the other arm; subsequent doses, if administered, continue toalternate in this manner. In some embodiments, the multiple doses areadministered as follows: a first dose is administered in one thigh, andsecond dose is administered in the other thigh; subsequent doses, ifadministered, continue to alternate in this manner. In some embodiments,the multiple doses are administered as follows: a first dose isadministered in one thigh, and second dose is administered in one arm;subsequent doses if administered can alternate in any combination thatis safe and efficacious for the subject. In some embodiments, themultiple doses are administered as follows: a first dose is administeredin one thigh and one arm, and second dose is administered in the otherarm and the other thigh; subsequent doses if administered can alternatein any combination that is safe and efficacious for the subject.

In some embodiments, the subject receives, via intradermal injection, avaccine composition comprising a total of six cell lines (e.g.,NCI-H460, NCI-H520, DMS 53, LK-2, NCI-H23, and A549 or other 6-cell linecombinations described herein) in one, two or more separate cocktails,each cocktail comprising one or a mixture two or more of the 6-celllines. In some embodiments, the subject receives, via intradermalinjection, a vaccine composition comprising a mixture of three celllines (e.g., three of NCI-H460, NCI-H520, DMS 53, LK-2, NCI-H23, andA549 or three cell lines from other 6-cell line combinations describedherein). In some embodiments, the subject receives, via intradermalinjection to the arm (e.g., upper arm), a vaccine composition comprisinga mixture of three cell lines, comprising NCI-H460, NCI-H520, and A549;and the subject concurrently receives, via intradermal injection to theleg (e.g., thigh), a vaccine composition comprising a mixture of threecell lines, comprising DMS 53, LK-2, and NCI-H23.

Where an additional therapeutic agent is administered, the doses ormultiple doses may be administered via the same or different route asthe vaccine composition(s). By way of example, a composition comprisinga checkpoint inhibitor is administered in some embodiments viaintravenous injection, and the vaccine composition is administered viaintradermal injection. In some embodiments, cyclophosphamide isadministered orally, and the vaccine composition is administeredintradermally.

Regimens

The vaccine compositions according to the disclosure may be administeredat various administration sites on a subject, at various times, and invarious amounts. The efficacy of a tumor cell vaccine may be impacted ifthe subject's immune system is in a state that is permissible to theactivation of antitumor immune responses. The efficacy may also thusimpacted if the subject is undergoing or has received radiation therapy,chemotherapy or other prior treatments. In some embodiments, thisrequires that the immunosuppressive elements of the immune system areinhibited while the activation and effector elements are fullyfunctional. In addition to the immunosuppressive factors describedherein, other elements that suppress antitumor immunity include, but arenot limited to, T regulatory cells (Tregs) and checkpoint molecules suchas CTLA-4, PD-1 and PD-L1.

In some embodiments, timing of the administration of the vaccinerelative to previous chemotherapy and radiation therapy cycles is set inorder to maximize the immune permissive state of the subject's immunesystem prior to vaccine administration. The present disclosure providesmethods for conditioning the immune system with one or low doseadministrations of a chemotherapeutic agent such as cyclophosphamideprior to vaccination to increase efficacy of whole cell tumor vaccines.In some embodiments, metronomic chemotherapy (e.g., frequent, low doseadministration of chemotherapy drugs with no prolonged drug-free break)is used to condition the immune system. In some embodiments, metronomicchemotherapy allows for a low level of the drug to persist in the blood,without the complications of toxicity and side effects often seen athigher doses. By way of example, administering cyclophosphamide tocondition the immune system includes, in some embodiments,administration of the drug at a time before the receipt of a vaccinedose (e.g., 15 days to 1 hour prior to administration of a vaccinecomposition) in order to maintain the ratio of effector T cells toregulatory T cells at a level less than 1.

In some embodiments, a chemotherapy regimen (e.g., myeloablativechemotherapy, cyclophosphamide, and/or fludarabine regimen) may beadministered before some, or all of the administrations of the vaccinecomposition(s) provided herein. Cyclophosphamide (CYTOXAN™, NEOSAR™) isa well-known cancer medication that interferes with the growth andspread of cancer cells in the body. Cyclophosphamide may be administeredas a pill (oral), liquid, or via intravenous injection. Numerous studieshave shown that cyclophosphamide can enhance the efficacy of vaccines.(See, e.g., Machiels et al., Cancer Res., 61:3689, 2001; Greten, T. F.,et al., J. Immunother., 2010, 33:211; Ghiringhelli et al., CancerImmunol. Immunother., 56:641, 2007; Ge et al., Cancer Immunol.Immunother., 61:353, 2011; Laheru et al., Clin. Cancer Res., 14:1455,2008; and Borch et al., Oncolmmunol, e1207842, 2016). “Low dose”cyclophosphamide as described herein, in some embodiments, is effectivein depleting Tregs, attenuating Treg activity, and enhancing effector Tcell functions. In some embodiments, intravenous low dose administrationof cyclophosphamide includes 40-50 mg/kg in divided doses over 2-5 days.Other low dose regimens include 1-15 mg/kg every 7-10 days or 3-5 mg/kgtwice weekly. Low dose oral administration, in accordance with someembodiments of the present disclosure, includes 1-5 mg/kg per day forboth initial and maintenance dosing. Dosage forms for the oral tabletare 25 mg and 50 mg. In some embodiments, cyclophosphamide isadministered as an oral 50 mg tablet for the 7 days leading up to thefirst and optionally each subsequent doses of the vaccine compositionsdescribed herein.

In some embodiments, cyclophosphamide is administered as an oral 50 mgtablet on each of the 7 days leading up to the first, and optionally oneach of the 7 days preceding each subsequent dose(s) of the vaccinecompositions. In another embodiment, the patient takes or receives anoral dose of 25 mg of cyclophosphamide twice daily, with one dose beingthe morning upon rising and the second dose being at night before bed, 7days prior to each administration of a cancer vaccine cocktail or unitdose. In certain embodiments, the vaccine compositions are administeredintradermally multiple times over a period of years. In someembodiments, a checkpoint inhibitor is administered every two weeks orevery three weeks following administration of the vaccinecomposition(s).

In another embodiment, the patient receives a single intravenous dose ofcyclophosphamide of 200, 250, 300, 500 or 600 mg/m² at least one dayprior to the administration of a cancer vaccine cocktail or unit dose ofthe vaccine composition. In another embodiment, the patient receives anintravenous dose of cyclophosphamide of 200, 250, 300, 500 or 600 mg/m²at least one day prior to the administration vaccine dose number 4, 8,12 of a cancer vaccine cocktail or unit dose. In another embodiment, thepatient receives a single dose of cyclophosphamide at 1000 mg/kg as anintravenous injection at least one hour prior to the administration of acancer vaccine cocktail or unit dose. In some embodiments, an oral highdose of 200 mg/kg or an IV high dose of 500-1000 mg/m² ofcyclophosphamide is administered.

The administration of cyclophosphamide can be via any of the following:oral (e.g., as a capsule, powder for solution, or a tablet); intravenous(e.g., administered through a vein (IV) by injection or infusion);intramuscular (e.g., via an injection into a muscle (IM));intraperitoneal (e.g., via an injection into the abdominal lining (IP));and intrapleural (e.g., via an injection into the lining of the lung).

In some embodiments, immunotherapy checkpoint inhibitors (e.g.,anti-CTLA4, anti-PD-1 antibodies such as pembrolizumab, and nivolumab,anti-PDL1 such as durvalumab) may be administered before, concurrently,or after the vaccine composition. In certain embodiments, pembrolizumabis administered 2 mg/kg every 3 weeks as an intravenous infusion over 60minutes. In some embodiments, pembrolizumab is administered 200 mg every3 weeks as an intravenous infusion over 30 minutes. In some embodimentspembrolizumab is administered 400 mg every 6 weeks as an intravenousinfusion over 30 minutes. In some embodiments, durvalumab isadministered 10 mg/kg every two weeks. In some embodiments, nivolumab isadministered 240 mg every 2 weeks (or 480 mg every 4 weeks). In someembodiments, nivolumab is administered 1 mg/kg followed by ipilimumab onthe same day, every 3 weeks for 4 doses, then 240 mg every 2 weeks (or480 mg every 4 weeks). In some embodiments, nivolumab is administered 3mg/kg followed by ipilimumab 1 mg/kg on the same day every 3 weeks for 4doses, then 240 mg every 2 weeks (or 480 mg every 4 weeks). In someembodiments, nivolumab is administered or 3 mg/kg every 2 weeks.

In some embodiments, durvalumab or pembrolizumab is administered every2, 3, 4, 5, 6, 7 or 8 weeks for up to 8 administrations and then reducedto every 6, 7, 8, 9, 10, 11 or 12 weeks as appropriate.

In other embodiments, the present disclosure provides that PD-1 andPD-L1 inhibitors are administered with a fixed dosing regimen (i.e., notweight-based). In non-limiting examples, a PD-1 inhibitor isadministered weekly or at weeks 2, 3, 4, 6 and 8 in an amount between100-1200 mg. In non-limiting examples, a PD-L1 inhibitor is administeredweekly or at weeks 2, 3, 4, 6 and 8 in an mount between 250-2000 mg.

In some embodiments, a vaccine composition or compositions as describedherein is administered concurrently or in combination with a PD-1inhibitor dosed either Q1W, Q2W, Q3W, Q4W, Q6W, or Q8W, between 100 mgand 1500 mg fixed or 0.5 mg/kg and 15 mg/kg based on weight. In anotherembodiment, a vaccine composition or compositions as described herein isadministered concurrently in combination with PD-L1 inhibitor dosedeither Q2W, Q3W, or Q4W between 250 mg and 2000 mg fixed or 2 mg/kg and30 mg/kg based on weight. In other embodiments, the aforementionedregimen is administered but the compositions are administered in shortsuccession or series such that the patient receives the vaccinecomposition or compositions and the checkpoint inhibitor during the samevisit.

The plant Cannabis sativa L. has been used as an herbal remedy forcenturies and is an important source of phytocannabinoids. Theendocannabinoid system (ECS) consists of receptors, endogenous ligands(endocannabinoids) and metabolizing enzymes, and plays a role indifferent physiological and pathological processes. Phytocannabinoidsand synthetic cannabinoids can interact with the components of ECS orother cellular pathways and thus may affect the development orprogression of diseases, including cancer. In cancer patients,cannabinoids can be used as a part of palliative care to alleviate pain,relieve nausea and stimulate appetite. In addition, numerous cellculture and animal studies have demonstrated antitumor effects ofcannabinoids in various cancer types. (For a review, see Daris, B., etal., Bosn. J. Basic. Med. Sci., 19(1):14-23 (2019).) Phytocannabinoidsare a group of C21 terpenophenolic compounds predominately produced bythe plants from the genus Cannabis. There are several differentcannabinoids and related breakdown products. Among these aretetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN),cannabichromene (CBC), Δ8-THC, cannabidiolic acid (CBDA), cannabidivarin(CBDV), and cannabigerol (CBG).

In certain embodiments of the present disclosure, use of allphytocannabinoids is stopped prior to or concurrent with theadministration of a Treg cell inhibitor such as cyclophosphamide, and/oris otherwise stopped prior to or concurrent with the administration of avaccine composition according to the present disclosure. In someembodiments, where multiple administrations of cyclophosphamide orvaccine compositions occur, the cessation optionally occurs prior to orconcurrent with each administration. In certain embodiments, use ofphytocannabinoids is not resumed until a period of time after theadministration of the vaccine composition(s). For example, abstainingfrom cannabinoid administration for at least 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 days prior to administration and at least 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 days after administration of cyclophosphamide or a vaccine doseis contemplated.

In some embodiments, patients will receive the first dose of the vaccinewithin 6-12 weeks after completion of chemotherapy. High dosechemotherapy used in cancer treatment ablates proliferating cells anddepletes immune cell subsets. Upon completion of chemotherapy, theimmune system will begin to reconstitute. The time span for T cells torecur is roughly 2-3 weeks. Because T cells are an immunological cellsubset targeted for activation, in some embodiments, the cancer vaccineis administered within a window where there are sufficient T cells toprime, yet the subject remains lymphopenic. This environment, in whichthere are less cells occupying the niche will allow the primed T cellsto rapidly divide, undergoing “homeostatic proliferation” in response toincreased availability of cytokines (e.g., IL7 and IL15). Thus, bydosing the vaccine at this window, the potential efficacy of embodimentsof the cancer vaccine platform as described herein is maximized to allowfor the priming of antigen specific T cells and expansion of the vaccineassociated T cell response.

Methods of Selecting Cell Lines and Preparing Vaccines

Cell Line Selection

For a given cancer or in instances where a patient is suffering frommore than one cancer, a cell line or combination of cell lines isidentified for inclusion in a vaccine composition based on severalcriteria. In some embodiments, selection of cell lines is performedstepwise as provided below. Not all cancer indications will require allof the selection steps and/or criteria.

Step 1. Cell lines for each indication are selected based on theavailability of RNA-seq data such as for example in the Cancer Cell LineEncyclopedia (CCLE) database. RNA-seq data allows for the identificationof candidate cell lines that have the potential to display the greatestbreadth of antigens specific to a cancer indication of interest andinforms on the potential expression of immunosuppressive factors by thecell lines. If the availability of RNA-seq data in the CCLE is limited,RNA-seq data may be sourced from the European Molecular BiologyLaboratory-European Bioinformatics Institute (EMBL-EBI) database orother sources known in the art. In some embodiments, potentialexpression of a protein of interest (e.g., a TAA) based on RNA-seq datais considered “positive” when the RNA-seq value is >0.

Step 2. For all indications, cell lines derived from metastatic sitesare prioritized to diversify antigenic breadth and to more effectivelytarget later-stage disease in patients with metastases. Cell linesderived from primary tumors are included in some embodiments to furtherdiversify breadth of the vaccine composition. The location of themetastases from which the cell line are derived is also considered insome embodiments. For example, in some embodiments, cell lines can beselected that are derived from lymph node, ascites, and liver metastaticsites instead of all three cell lines derived from liver metastaticsites.

Step 3. Cell lines are selected to cover a broad range ofclassifications of cancer types. For example, tubular adenocarcinoma isa commonly diagnosed classification of gastric cancer. Thus, numerouscell lines may be chosen matching this classification. For indicationswhere primary tumor sites vary, cell lines can be selected to meet thisdiversity. For example, for small cell carcinoma of the head and neck(SCCHN), cell lines were chosen, in some embodiments, to cover tumorsoriginating from the oral cavity, buccal mucosa, and tongue. Theseselection criteria enable targeting a heterogeneous population ofpatient tumor types. In some embodiments, cell lines are selected toencompass an ethnically diverse population to generate a cell linecandidate pool derived from diverse histological and ethnicalbackgrounds.

Step 4. In some embodiments, cell lines are selected based on additionalfactors. For example, in metastatic colorectal cancer (mCRC), cell linesreported as both microsatellite instable high (MSI-H) and microsatellitestable (MSS) may be included. As another example, for indications thatare viral driven, cell lines encoding viral genomes may be excluded forsafety and/or manufacturing complexity concerns.

Step 5. In some embodiments, cell lines are selected to cover a varyingdegree of genetic complexity in driver mutations orindication-associated mutations. Heterogeneity of cell line mutationscan expand the antigen repertoire to target a larger population withinpatients with one or more tumor types. By way of example, breast cancercell lines can be diversified on deletion status of Her2, progesteronereceptor, and estrogen receptor such that the final unit dose includestriple negative, double negative, single negative, and wild typecombinations. Each cancer type has a complex genomic landscape and, as aresult, cell lines are selected for similar gene mutations for specificindications. For example, melanoma tumors most frequently harboralterations in BRAF, CDKN2A, NRAS and TP53, therefore selected melanomacell lines, in some embodiments, contain genetic alterations in one ormore of these genes.

Step 6. In some embodiments, cell lines are further narrowed based onthe TAA, TSA, and/or cancer/testis antigen expression based on RNA-seqdata. An antigen or collection of antigens associated with a particulartumor or tumors is identified using search approaches evident to personsskilled in the art (See, e.g., such as www.ncbi.nlm.nih.gov/pubmed/, andclinicaltrials.gov). In some embodiments, antigens can be included ifassociated with a positive clinical outcome or identified ashighly-expressed by the specific tumor or tumor types while expressed atlower levels in normal tissues.

Step 7. After Steps 1 through 6 are completed, in some embodiments, thelist of remaining cell line candidates are consolidated based on cellculture properties and considerations such as doubling time, adherence,size, and serum requirements. For example, cell lines with a doublingtime of less than 80 hours or cell lines requiring media serum (FBS,FCS)<10% can be selected. In some embodiments, adherent or suspensioncell lines that do not form aggregates can be selected to ensure propercell count and viability.

Step 8. In some embodiments, cell lines are selected based on theexpression of immunosuppressive factors (e.g., based on RNA-seq datasourced from CCLE or EMBL as described in Step 1).

In some embodiments, a biopsy of a patient's tumor and subsequent TAAexpression profile of the biopsied sample will assist in the selectionof cell lines. Embodiments of the present disclosure therefore provide amethod of preparing a vaccine composition comprising the steps ofdetermining the TAA expression profile of the subject's tumor; selectingcancer cell lines; modifying cancer cell lines; and irradiating celllines prior to administration to prevent proliferation afteradministration to patients.

Preparing Vaccine Compositions

In certain embodiments, after expansion in manufacturing, all of thecells in a modified cell line are irradiated, suspended, andcryopreserved. In some embodiments, cells are irradiated 10,000 cGy.According to some embodiments, cells are irradiated at 7,000 to 15,000cGy. According to some embodiments, cells are irradiated at 7,000 to15,000 cGy.

In certain embodiments, each vial contains a volume of 120±10 μL(1.2×10⁷ cells). In some embodiments, the total volume injected per siteis 300 μL or less. In some embodiments, the total volume injected persite is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, or 300 μL. Where, for example, the total volume injected is 300 μL,the present disclosure provides, in some embodiments that 3×100 μLvolumes, or 2×150 μL, are injected, for a total of 300 μL.

In some embodiments, the vials of the component cell lines are stored inthe liquid nitrogen vapor phase until ready for injection. In someembodiments, each of the component cell lines are packaged in separatevials.

As described herein, prior to administration, in some embodiments thecontents of two vials are removed by needle and syringe and are injectedinto a third vial for mixing. In some embodiments, this mixing isrepeated for each cocktail. In other embodiments, the contents of sixvials are divided into two groups—A and B, where the contents of threevials are combined or mixed, optionally into a new vial (A), and thecontents of the remaining three vials are combined or mixed, optionallyinto a new vial (B).

In certain embodiments, the cells will be irradiated prior tocryopreservation to prevent proliferation after administration topatients. In some embodiments, cells are irradiated at 7,000 to 15,000cGy in order to render the cells proliferation incompetent.

In some embodiments, cell lines are grown separately and in the samegrowth culture media. In some embodiments, cell lines are grownseparately and in different cell growth culture media.

Xeno-Free Conversion of Whole Tumor Cell Vaccine Component Cell Lines

Analysis of antibody responses in subjects treated with a whole tumorcell vaccine has suggested a negative correlation between survival andthe development of IgG antibody responses to the bovine α-Gal antigen.(See Xia et al., Cell Chem Biol 23(12):1515-1525 (2016)). This issignificant because most whole tumor cell vaccines are comprised oftumor cell lines that have been expanded and cryopreserved in mediacontaining fetal bovine serum (FBS), which contains the bovine α-Galantigen.

In some embodiments, to prevent the immune response to foreign antigensthat are present in FBS, the cell lines disclosed herein are adapted toxeno-free media composed of growth factors and supplements essential forcell growth that are from human source, prior to large scale cGMPmanufacturing. As used herein, the terms “adapting” and “converting” or“conversion” are used interchangeably to refer to transferring/changingcells to a different media as will be appreciated by those of skill inthe art. The xeno-free media formulation chosen can be, in someembodiments, the same across all cell lines or, in other embodiments,can be different for different cell lines. In some embodiments, themedia composition will not contain any non-human materials and caninclude human source proteins as a replacement for FBS alone, or acombination of human source proteins and human source recombinantcytokines and growth factors (e.g., EGF). Additionally, the xeno-freemedia compositions can, in some embodiments, also contain additionalsupplements (e.g., amino acids, energy sources) that enhance the growthof the tumor cell lines. The xeno-free media formulation will beselected for its ability to maintain cell line morphology and doublingtime no greater than twice the doubling time in FBS and the ability tomaintain expression of transgenes comparable to that in FBS.

A number of procedures may be instituted to minimize the possibility ofinducing IgG, IgA, IgE, IgM and IgD antibodies to bovine antigens. Theseinclude but are not limited to: cell lines adapted to growth inxeno-free media; cell lines grown in FBS and placed in xeno-free mediafor a period of time (e.g., at least three days) prior to harvest; celllines grown in FBS and washed in xeno-free media prior to harvest andcryopreservation; cell lines cryopreserved in media containing Buminate(a USP-grade pharmaceutical human serum albumin) as a substitute forFBS; and/or cell lines cryopreserved in a medial formulation that isxeno-free, and animal-component free (e.g., CryoStor). In someembodiments, implementation of one or more of these procedures mayreduce the risk of inducing anti-bovine antibodies by removing thebovine antigens from the vaccine compositions.

According to one embodiment, the vaccine compositions described hereindo not comprise non-human materials. In some embodiments, the cell linesdescribed herein are formulated in xeno-free media. Use of xeno-freemedia avoids the use of immunodominant xenogeneic antigens and potentialzoonotic organisms, such as the BSE prion. By way of example, followinggene modification, the cell lines are transitioned to xeno-free mediaand are expanded to generate seed banks. The seed banks arecryopreserved and stored in vapor-phase in a liquid nitrogen cryogenicfreezer.

Exemplary xeno-free conversions are provided herein for a NSCLC and GBMvaccine preparations.

In Vitro Assays

The ability of allogeneic whole cell cancer vaccines such as thosedescribed herein, to elicit anti-tumor immune responses, and todemonstrate that modifications to the vaccine cell lines enhancevaccine-associated immune responses, can be modelled with in vitroassays. Without being bound by any theory, the genetic modificationsmade to the vaccine cell line components augment adaptive immuneresponses through enhancing dendritic cell (DC) function in the vaccinemicroenvironment. The potential effects of expression of TAAs,immunosuppressive factors, and/or immunostimulatory factors can bemodelled in vitro, for example, using flow cytometry-based assays andthe IFNγ ELISpot assay.

In some embodiments, to model the effects of modifications to thevaccine cell line components in vitro, DCs are derived from monocytesisolated from healthy donor peripheral blood mononuclear cells (PBMCs)and used in downstream assays to characterize immune responses in thepresence or absence of one or more immunostimulatory orimmunosuppressive factors. The vaccine cell line components arephagocytized by donor-derived immature DCs during co-culture with theunmodified parental vaccine cell line (control) or the modified vaccinecell line components. The effect of modified vaccine cell linecomponents on DC maturation, and thereby subsequent T cell priming, canbe evaluated using flow cytometry to detect changes in markers of DCmaturation such as CD40, CD83, CD86, and HLA-DR. Alternatively, theimmature DCs are matured after co-culture with the vaccine cell linecomponents, the mature DCs are magnetically separated from the vaccinecell line components, and then co-cultured with autologous CD14-PBMCsfor 6 days to mimic in vivo presentation and stimulation of T cells.IFNγ production, a measurement of T cell stimulatory activity, ismeasured in the IFNγ ELISpot assay or the proliferation andcharacterization of immune cell subsets is evaluated by flow cytometry.In the IFNγ ELISpot assay, PBMCs are stimulated with autologous DCsloaded with the unmodified parental vaccine cell line components toassess potential responses against unmodified tumor cells in vivo.

The IFNγ ELISpot assay can be used to evaluate the potential of theallogenic vaccine to drive immune responses to clinically relevant TAAsexpressed by the vaccine cell lines. To assess TAA-specific responses inthe IFNγ ELISpot assay, following co-culture with DCs, the PBMCs arestimulated with peptide pools comprising known diverse MHC-I epitopesfor TAAs of interest. In various embodiments, the vaccine compositionmay comprise 3 cell lines that induce IFNγ responses to at least 3, 4,5, 6, 7, 8, 9, 10, or 11 non-viral antigens, or at least 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% of the antigens evaluated for an IFNγresponse. In some embodiments, the vaccine composition may be a unitdose of 6 cell lines that induce IFNγ responses to at least 5, 6, 7, 8,9, 10 or 11 non-viral antigens, or at least 60%, 70%, 80%, 90%, or 100%of the antigens evaluated for an IFNγ response.

In Vivo Mouse Models

Induction of antigen specific T cells by the allogenic whole cellvaccine can be modeled in vivo using mouse tumor challenge models. Thevaccines provided in embodiments herein may not be administered directlyto mouse tumor model due to the diverse xenogeneic homology of TAAsbetween mouse and human. However, a murine homolog of the vaccines canbe generated using mouse tumor cell lines. Some examples of additionalimmune readouts in a mouse model are: characterization of humoral immuneresponses specific to the vaccine or TAAs, boosting of cellular immuneresponses with subsequent immunizations, characterization of DCtrafficking and DC subsets at draining lymph nodes, evaluation ofcellular and humoral memory responses, reduction of tumor burden, anddetermining vaccine-associated immunological changes in the TME, such asthe ratio of tumor infiltrating lymphocytes (TILs) to Tregs. Standardimmunological methods such as ELISA, IFNγ ELISpot, and flow cytometrywill be used.

Kits

The vaccine compositions described herein may be used in the manufactureof a medicament, for example, a medicament for treating or prolongingthe survival of a subject with cancer, e.g., lung cancer, non-small celllung cancer (NSCLC), small cell lung cancer (SCLC), prostate cancer,glioblastoma, colorectal cancer, breast cancer including triple negativebreast cancer (TNBC), bladder or urinary tract cancer, squamous cellhead and neck cancer (SCCHN), liver hepatocellular (HCC) cancer, kidneyor renal cell carcinoma (RCC) cancer, gastric or stomach cancer, ovariancancer, esophageal cancer, testicular cancer, pancreatic cancer, centralnervous system cancers, endometrial cancer, melanoma, and mesotheliumcancer.

Also provided are kits for treating or prolonging the survival of asubject with cancer containing any of the vaccine compositions describedherein, optionally along with a syringe, needle, and/or instructions foruse. Articles of manufacture are also provided, which include at leastone vessel or vial containing any of the vaccine compositions describedherein and instructions for use to treat or prolong the survival of asubject with cancer. Any of the vaccine compositions described hereincan be included in a kit comprising a container, pack, or dispensertogether with instructions for administration.

In some embodiments, provided herein is a kit comprising at least twovials, each vial comprising a vaccine composition (e.g., cocktail A andcocktail B), wherein each vial comprises at least 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more cell lines, wherein the cell lines are modified toinhibit or reduce production of one or more immunosuppressive factors,and/or express or increase expression of one or more immunostimulatoryfactors, and/or express a heterogeneity of tumor associated antigens, orneoantigens.

By way of example, a kit comprising 6 separate vials is provided,wherein each vial comprises one of the following cell lines: NCI-H460,NCI-H520, DMS 53, LK-2, NCI-H23, and A549. As another example, a kitcomprising 6 separate vials is provided, wherein each vial comprises oneof the following cell lines: DMS 53, DBTRG-05MG, LN-229, SF-126, GB-1,and KNS-60. As another example, a kit comprising 6 separate vials isprovided, wherein each vial comprises one of the following cell lines:DMS53, PC3, NEC8, NTERA-2c1-D1, DU-145, and LNCAP. As another example, akit comprising 6 separate vials is provided, wherein each vial comprisesone of the following cell lines: DMS 53, HCT-15, HuTu80, LS411N, HCT-116and RKO. As another example, a kit comprising 6 separate vials isprovided, wherein each vial comprises one of the following cell lines:DMS 53, OVTOKO, MCAS, TOV-112D, TOV-21G, and ES-2. As another example, akit comprising 6 separate vials is provided, wherein each vial comprisesone of the following cell lines: DMS 53, HSC-4, HO-1-N-1, DETROIT 562,KON, and OSC-20. As another example, a kit comprising 6 separate vialsis provided, wherein each vial comprises one of the following celllines: DMS 53, J82, HT-1376, TCCSUP, SCaBER, and UM-UC-3. As anotherexample, a kit comprising 6 separate vials is provided, wherein eachvial comprises one of the following cell lines: DMS 53, MKN-1, MKN-45,MKN-74, OCUM-1, and Fu97. As another example, a kit comprising 6separate vials is provided, wherein each vial comprises one of thefollowing cell lines: DMS 53, AU565, CAMA-1, HS-578T, MCF-7, and T-47D.As another example, a kit comprising 6 separate vials is provided,wherein each vial comprises one of the following cell lines: DMS 53,PANC-1, KP-3, KP-4, SUIT-2, and PSN1.

In some embodiments, provided herein is a kit comprising at least twovials, each vial comprising a vaccine composition (e.g., cocktail A andcocktail B), wherein each vial comprises at least three cell lines,wherein the cell lines are modified to reduce production or expressionof one or more immunosuppressive factors, and/or modified to increaseexpression of one or more immunostimulatory factors, and/or express aheterogeneity of tumor associated antigens, or neoantigens. The twovials in these embodiments together are a unit dose. Each unit dose canhave from about 5×10⁶ to about 5×10⁷ cells per vial, e.g., from about5×10⁶ to about 3×10⁷ cells per vial.

In some embodiments, provided herein is a kit comprising at least sixvials, each vial comprising a vaccine composition, wherein each vaccinecomposition comprises one cell line, wherein the cell line is modifiedto inhibit or reduce production of one or more immunosuppressivefactors, and/or modified to express or increase expression of one ormore immunostimulatory factors, and/or expresses a heterogeneity oftumor associated antigens, or neoantigens. Each of the at least sixvials in the embodiments provided herein can be a unit dose of thevaccine composition. Each unit dose can have from about 2×10⁶ to about50×10⁶ cells per vial, e.g., from about 2×10⁶ to about 10×10⁶ cells pervial.

In some embodiments, provided herein is a kit comprising separate vials,each vial comprising a vaccine composition, wherein each vaccinecomposition comprises one cell line, wherein the cell line is modifiedto inhibit or reduce production of one or more immunosuppressivefactors, and/or modified to express or increase expression of one ormore immunostimulatory factors, and/or expresses, a heterogeneity oftumor associated antigens, or neoantigens. Each of the vials in theembodiments provided herein can be a unit dose of the vaccinecomposition. Each unit dose can have from about 2×10⁶ to about 50×10⁶cells per vial, e.g., from about 2×10⁶ to about 10×10⁶ cells per vial.

In one exemplary embodiment, a kit is provide comprising two cocktailsof 3 cell lines each (i.e., total of 6 cell lines in 2 different vaccinecompositions) as follows: 8×10⁶ cells per cell line; 2.4×10⁷ cells perinjection; and 4.8×10⁷ cells total dose. In another exemplaryembodiment, 1×10⁷ cells per cell line; 3.0×10⁷ cells per injection; and6.0×10⁷ cells total dose is provided. In some embodiments, a vial of anyof the kits disclosed herein contains about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, or 1.0 mL of a vaccine composition of thedisclosure. In some embodiments, the concentration of cells in a vial isabout 5×10⁷ cells/mL to about 5×10⁸/cells mL.

The kits as described herein can further comprise needles, syringes, andother accessories for administration.

EXAMPLES Example 1: Reduction of HLA-G Expression in a HumanAdenocarcinoma Cell Line of the Lung Increases IFNγ Secretion in aCo-Culture with Peripheral Blood Mononuclear Cells (PBMC)

Aberrant expression of HLA-G by tumor cell is associated with tumorimmune escape, metastasis and poor prognosis. Ligation of HLA-G with itsreceptors ILT2 and ILT4 on DCs can promote immune tolerance and primingof T cells with an immunosuppressed phenotype. Reduction of HLA-Gexpression on cell line component of a whole cell vaccine could improveimmunogenicity in the VME.

Reduction of HLA-G Expression in Human Adenocarcinoma Cell Line

Human adenocarcinoma cell line RERF-LC-Ad1 was transduced withlentiviral particles expressing a short-hairpin ribonucleic acid (shRNA)specific for the knockdown of HLA-G (mature antisense sequence:TACAGCTGCAAGGACAACCAG) (SEQ ID NO: 23). Parental cells or cellstransduced with control (non-silencing) shRNA served as controls. HLA-Gexpression levels following shRNA mediated HLA-G knockdown wasdetermined by cytometry by staining with an APC-conjugated mousemonoclonal antibody human HLA-G (clone 87G) and then FACs sorted toenrich for the HLA-G low population. Modified and unmodified cells weredetached and stained with an APC-conjugated mouse monoclonal antibodyhuman HLA-G (clone 87G). After selection with puromycin to enrich forcells stable expressing the shRNA, cells were analyzed for expression ofHLA-G at mRNA level by quantitative polymerase chain reaction (qPCR) andat protein level by flow cytometry. For qPCR cells were lysed in Trizol,total RNA isolated and then transcribed into complementary DNA (cDNA).Relative HLA-G mRNA expression was quantified with specific-probes forHLA-G and PSMB4 (for normalization) using the MCt method. HLA-G mRNAexpression was reduced in cells stable transduced with shRNA for HLA-Gin comparison to parental (non-transduced) cells and cells transducedwith control (non-silencing) shRNA by at least 75% (FIG. 1A). HLA-Gexpression levels were following shRNA mediated HLA-G knockdown wasdetermined by flow cytometry. Modified and unmodified cells weredetached and stained with an APC-conjugated mouse monoclonal antibodyhuman HLA-G (clone 87G). Fluorescence (expression) intensity wascalculated as delta mean fluorescence intensity(ΔMFI=MFI_(anti-HLA-G)−MFI_(unstained)). HLA-G cell surface expressionwas reduced in in cells stable transduced with shRNA for HLA-G incomparison to parental (non-transduced) cells by 70% (FIG. 1B).

Increase of IFNγ Secretion in Mixed Lymphocyte Tumor Reaction (MLR)

PBMCs were isolated from blood of healthy donors and co-incubated withadenocarcinoma lung cancer cell lines, that were pre-treated withmitomycin C (0.4 μg/ml for 16 hours) to prevent tumor cell growth andproliferation, at a PBMC to tumor cell ratio of 10 to 1. Interleukin-2(IL2) was added on day 3 (and 7) of co-culture at differentconcentrations. On day 7 and/or 10 cell culture supernatant washarvested and IFNγ secretion was measured by ELISA. The increase of IFNγin the co-culture of PBMCs with tumor cells with reduced HLA-Gexpression was significant (p<0.01) compared to parental andnon-silencing tumor cells on day 10 (2way ANOVA with Sidak's multiplecomparisons test) (FIG. 2A). In addition, the significant increase ofIFNγ secretion was independent of the IL-2 concentration duringco-culture (p<0.0001, 2way ANOVA with Tukey's multiple comparisons test)(FIG. 2B).

Example 2: Reduction of CD47 Expression Increases Phagocytosis of TumorCell Lines by Antigen Presenting Cells and Enhances Immunogenicity

CD47 is a cell surface marker for “self” and thereby preventsimmunological responses against healthy cells. Primary tumor cells aswell as tumor cell lines can express high levels of CD47.

Reduction of CD47 Expression in Human Adenocarcinoma Cell Line

The human NSCLC cell lines A549, NCI-H460, and NCI-H520 wereelectroporated with a zinc finger nuclease (ZFN) pair specific for CD47targeting the following genomic DNA sequence:CACACAGGAAACTACacttgtGAAGTAACAGAATTA (SEQ ID NO: 27). Full-allelicknockout cells were identified by flow cytometry after staining withPE-conjugated anti-human CD47 monoclonal antibody (clone CC2C6) and thenFACS sorted to enrich for the CD47 negative population. Gene editing ofCD47 by ZFN resulted in greater than 99% reduction in CD47 expression bythe A549 (FIG. 3A), NCI-H460 (FIG. 3B), and NCI-H520 (FIG. 3C) celllines.

Reduction of CD47 Increases Phagocytosis of Tumor Cell Lines by AntigenPresenting Cells and Enhances Immunogenicity

The effect of reducing CD47 expression (CD47 KO) on phagocytosis andimmunogenicity was determined using the NCI-H520 cell line.Specifically, the effect of CD47 KO on phagocytosis by humanmonocyte-derived professional antigen presenting cells (APCs), both DCsand macrophages, was determined using a phagocytosis assay. Immuneresponses induced by NCI-H520 unmodified parental and CD47 KO evaluatedin the IFNγ ELISpot assay.

Generation of Human Dendritic Cells and Macrophages

Human immature dendritic cells (iDCs) and M1 macrophages (MDM) werederived from CD14⁺ cells isolated from healthy donor leukopaks (StemCellTechnologies, #70500) by magnetic separation according to themanufacturer's instructions. iDCs were generated by culturing CD14⁺cells in ImmunoCult™-ACF Dendritic Cell Medium (StemCell Technologies,#10986) in the presence of ImmunoCult™-ACF Dendritic CellDifferentiation Supplement (StemCell Technologies, #10988) according tothe manufacturers instructions. iDCs were harvested for use in thephagocytosis assay on Day 3 and on Day 6 for use in the IFNγ ELISpotassay. MDM were generated by culturing CD14⁺ cells in RPMI supplementedwith 10% FBS in the presence of 100 ng/mL GM-CSF (PeproTech,#300-03-100UG) for 7 days. To skew macrophages towards a M1 phenotype,on Day 7 the RPMI+10% FBS media was replaced with Macrophage-SFM (Gibco,#12065074) containing 20 ng/mL LPS (InvivoGen, #tlrl-3pelps) and 20ng/mL IFNγ (PeproTech, 300-02-100UG). MDM were harvested on Day 9 forthe phagocytosis assay.

Phagocytosis Assay

Unmodified parental and CD47 KO NCI-H520 cells were treated with 10μg/mL mitomycin C (MMC) for 2 hours and rested overnight prior tolabelling with 1 μM of CSFE (Invitrogen, #C34554) for 30 minutes at 37°C. iDC and MDM were co-cultured with the CSFE-labeled unmodifiedparental and CD47 KO NCI-H520 cells for 4 hours at 37° C. iDC and celllines were co-cultured at a 1:1 effector to target ratio in 96-welllow-adherence U bottom plates. MDM were co-cultured at a 1:4 effector totarget ratio in 96-well plates. Following the 4 hour incubation, theco-cultures were surface stained with LIVE/DEAD Aqua (Molecular Probes,#L23105), αCD45-PE-Cy7 (BD Biosciences, clone HI30), and αCD11c-BV605(BD Biosciences, clone B-Iy6) for iDCs or αCD11b-BV421 (BD Biosciences,clone ICRF44) for MDM. Flow cytometry data was analyzed using FlowJo(FlowJo LLC). MDM phagocytosis was defined as the percentage of live,CD45⁺, CD11b⁺ cells that were also CFSE (FITC) positive by flowcytometry. iDC phagocytosis was defined as the percent of live, CD45⁺,CD11c⁺ cells that were also CFSE (FITC⁺) positive by flow cytometry. MDMand iDC that were not co-cultured with the unmodified parental or CD47KO NCI-H520 cells served as controls.

IFNγ ELISpot Assay

Unmodified parental and CD47 KO NCI-H520 cells were x-ray irradiated at100 Gy (Rad Source 1800 Q) 24 hours prior to loading of iDCs. To loadiDCs, irradiated unmodified parental and CD47 KO NCI-H520 (ATCC HTB-182)were co-cultured with iDCs at a 1:1 ratio for 24 hours in the presenceof 25 μg/mL of Keyhole Limpet Hemocyanin (KLH) (Calbiochem #374807) and1 μg/mL soluble CD40L (sCD40L) (PeproTech, #AF31002100UG). Tumor cellloaded iDCs were than matured overnight by the addition of 100 IU/mLIFNγ (PeproTech, 300-02-100UG), 10 ng/mL LPS (InvivoGen, #tlrl-3pelps)and 2.5 μg/mL Resiquimod (R848) (InvivoGen, #tlrl-3r848). Mature DCs(mDCs) were labelled with αCD45-PE (BD Biosciences, clone H130) andmagnetically separated from the co-culture using the EasySep™ ReleaseHuman PE Positive Selection Kit (StemCell Technologies, #17654)according to manufacturers instructions. Isolated mDCs were thenco-cultured with autologous CD14⁻ PBMCs for 6 days at a 1:10 DC to PBMCratio. For the IFNγ ELISpot assay (MabTech, 3420-4APT-10), CD14⁻ PBMCswere isolated from co-culture with mDCs and stimulated with unmodifiedparental NCI-H520 loaded mDCs for 24 hours. IFNγ spot forming units(SFU) were detected following the manufacturers instructions, counted(S6 Core Analyzer, ImmunoSpot), and expressed as the number of SFU/10⁶PBMCs above that of the controls.

Increased Phagocytosis of the NCI-H520 CD47KO Cell Line by MonocyteDerived Dendritic Cells and Macrophages

Reduction of CD47 increased phagocytosis by MDM derived from 2 healthydonors by an average of 1.6-fold (11.1±1.9% live/CD45⁺/CD11b⁺/CFSE⁺)relative to phagocytosis of the unmodified parental cell line (7.0±1.2%live/CD45⁺/CD11b⁺/CFSE⁺). Reduction of CD47 also increased phagocytosisby iDC derived from 2 healthy donors by an average of 2.2-fold(11.9±2.3% live/CD45⁺/CD11c⁺/CFSE⁺) relative to phagocytosis of theunmodified parental cell line (5.5±3.4% live/CD45⁺/CD11c⁺/CFSE⁺) (FIG.4A).

Reduction of CD47 Improves Immunogenicity of a Human Squamous Tumor CellLine

IFNγ responses by ELISpot were 1.9-fold higher when autologous PBMCswere co-cultured with DCs loaded with CD47 KO cells (9,980±903 SFU)relative to DCs loaded with the unmodified parental, CD47 positive cells(5,253±109 SFU) (p=0.007, Student's T-test) (n=3) (FIG. 4B).

Example 3: Reduction of Programmed Cell Death Ligand 1 Expression

Binding of PD1 on DCs to PDL1 (CD274) on tumor cells can suppress DCfunction and potentially reduce priming of inflammatory (Th₁) T cellsand promote the priming of immunosuppressive (Th₂) T cells.

PDL1 expression by the NSCLC cell line NCI-H460 was reduced usingzinc-finger mediated gene editing. The cell line was electroporated withDNA plasmids coding for a zinc finger nuclease (ZFN) pair specific forPD-L1 targeting the following genomic DNA sequence:CCAGTCACCTCTGAACATGaactgaCATGTCAGGCTGAGGGCT (SEQ ID NO: 28).Full-allelic knockout cells were identified by flow cytometry afterstaining with PE-conjugated anti-human CD274 monoclonal antibody (cloneMIH1) and then FACS sorted. Gene editing of PD-L1 by ZFNs resulted ingreater than 99% PD-L1 negative NCI-H460 cells after sorting (FIG. 5).

Example 4: Reduction of Bone Marrow Stromal Cell Antigen 2 (bst2)Expression

BST2 is a cell surface marker on primary tumor cells and tumor celllines that inhibits cytokine production (type I interferons) throughinteraction with ILT7 (CD85g) on plasmacytoid dendritic cells.

The reduction of BST2 expression by the NCI-H2009 cell line wascompleted using ZFN mediated gene editing. The cell line waselectroporated with DNA plasmids coding for a ZFN pair specific for BST2targeting the following genomic DNA sequence:CCTAATGGCTTCCCTGGATgcagagAAGGCCCAAGGACAAAAG (SEQ ID NO: 34).Full-allelic knockout cells were identified by flow cytometry afterstaining with BV421-conjugated anti-human BST2 monoclonal antibody(clone HM1.24). Gene editing of BST2 by ZFNs resulted in 98.5% reductionin BST2 expression by NCI-H2009 cells (FIG. 6). The BST2 positivefraction of BST2-ZFN treated NCI-H2009 cells can subsequently be FACSsorted to purity.

Example 5: Reduction of TGFβ1 and/or TGFβ2 Secretion in Lung Cancer CellLines

TGFβ1 and TGFβ2 are highly immunosuppressive molecules secreted by tumorcells to evade immune surveillance. This example describes the procedureto generate lung cancer cell lines with reduced or without secretion ofTGFβ1 and TGFβ2 and how the changes in secretion were verified.

Cell Lines, Culture and Selection

The lung cancer cell lines NCI-H460 (ATCC HTB-177), DMS 53 (ATCCCRL-2062), NCI-H520 (ATCC HTB-182), A549 (ATCC CCL-185), NCI-H2023 (ATCCCRL-5912), NCI-H23 (ATCC CRL-5800), and NCI-H1703 (ATCC CRL-5889) wereobtained from ATCC and cultured according to ATCC recommendations. LK-2(JCRB0829) was obtained from the Japanese Collection of ResearchBiosources Cell Bank (JCRB) and cultured according to JCRBrecommendations. For mammalian cell line selection after lentiviraltransduction puromycin and blasticidin in concentrations ranging from 2to 8 μg/mL were used for selection and maintenance.

shRNA Mediated Knockdown of TGFβ1 and TGFβ2

The cell lines NCI-H460, DMS 53, and NCI-H520, A549, NCI-H2023, NCI-H23,LK-2, and NCI-H1703 were transduced with lentiviral particles expressingshort-hairpin ribonucleic acid (shRNA) specific for the knockdown ofTGFβ1 (shTGFβ1, mature antisense sequence: TTTCCACCATTAGCACGCGGG (SEQ IDNO: 25)) and TGFβ2 (shTGFβ2, mature antisense sequence:AATCTGATATAGCTCAATCCG (SEQ ID NO: 24)). Cells transduced with controlshRNA (NS) or parental unmodified cell lines served as controls. Afterantibiotic selection to enrich for cells stabling expressing shRNA(s),cells were analyzed for TGFβ1 and TGFβ2 secretion.

Knockout of TGFβ1 and TGFβ2

Knockout of TGFβ1 and TGFβ2 was completed using CRISPR-Cas9 and ZFNapproaches. For CRISPR-Cas9 knockouts, the NCI-H460 and NCI-H520 celllines were electroporated with plasmid DNA coding for Cas9 and guide RNAspecific for TGFβ2 targeting the following gDNA sequence:GCTTGCTCAGGATCTGCCCG (SEQ ID NO: 29) or control guide RNA targeting thesequence: GCACTACCAGAGCTAACTCA (SEQ ID NO: 30). Full-allelic knockoutclones were screened for secretion of TGFβ1 and TGFβ2 by ELISA. ForZFN-mediated knockout, the NCI-H460 cell line was electroporated withRNA coding for zinc finger nuclease (ZFN) pairs specific for TGFβ1targeting the following genomic DNA (gDNA) sequence:CTCGCCAGCCCCCCGagccaGGGGGAGGTGCCGCCCGG (SEQ ID NO: 31) and for TGFβ2targeting the following gDNA sequence:AGCTCACCAGTCCCCCAGAagactaTCCTGAGCCCGAGGAAGTC (SEQ ID NO: 32).Full-allelic knockout clones were screened by genomic DNA sequencing ofexpanded single cells and then analyzed for TGFβ1 and TGFβ2 secretion.

TGFβ1 and TGFβ2 Secretion Assay

TGFβ1 and TGFβ2 knockdown or knockout cells and unmodified or controlmodified parental cells were plated at 8.33×10⁴ cells/well in a 24-wellplated in regular growth medium (RPMI containing 10% FBS). Twenty-fourhours after plating, adherent cells were thoroughly washed to remove FBSand culture was continued in RPMI+5% CTS. Forty-eight hours after mediareplacement, the cell culture supernatant was harvested, and stored at−70° C. until TGFβ1 and TGFβ2 secretion assays were initiated accordingto the manufacturer's instructions (DB100B and DB250, R&D Systems).TGFβ1 and TGFβ2 secretion levels are expressed as pg/10⁶ cells/24 hours.The lower limit of quantification of human TGFβ1 and TGFβ2 are 15.4μg/mL (92.4 μg/10⁶ cells/24 hours) and 7.0 μg/mL (42.0 μg/10⁶ cells/24hours), respectively. The lower limit of quantification of the ELISAassay was used to approximate the percent reduction of TGFβ1 or TGFβ2relative to the unmodified parental cell line shRNA when the modifiedcell lines secreted levels of TGFβ1 or TGFβ2 below the lower limit ofquantification of the assay. In cases where TGFβ1 or TGFβ2 secretionwere below the lower limit of quantification, the lower limit ofquantification was used to determine statistical significance at the nfor which the assay was completed.

Reduction of TGFβ1 and TGFβ2 Secretion in NCI-H460 Cells

Knockdown of TGFβ1 in NCI-H460 reduced TGFβ1 secretion by 62%.Similarly, knockdown of TGFβ2 in NCI-H460 reduced TGFβ2 secretion by84%. The combined knockdown of TGFβ1 and TGFβ2 in NCI-H460 reduced TGFβ1secretion by 57% and TGFβ2 secretion by >98% (Table 26) (FIG. 7A).Clones derived from Cas9 mediated knockout using TGFβ2 specific guideRNA in NCI-H460 cells demonstrated clones did not secrete TGFβ2 (>99%reduction) above the lower limit of detect compared clones from NCI-H460treated with control guide RNA (3686±1478 μg/10⁶ cells/24 hours) (FIG.7B). Clones derived from NCI-H460 treated with TGFβ1 specific ZFN pairdid not secrete TGFβ1 above the lower limit of detection of the assaycompared to clones from NCI-H460 treated with TGFβ2 specific ZFN pair.Clones derived from NCI-H460 treated with TGFβ2 specific ZFN pair didnot secrete TGFβ2 above the lower limit of detection in contrast toclones from NCI-H460 treated with TGFβ1 specific ZFN pair. Clonesderived from NCI-H460 treated with TGFβ1 specific ZFN pair and withTGFβ2 specific ZFN pair did not secrete TGFβ1 or TGFβ2 above the lowerlimit of detection (FIG. 7C).

Knockdown of TGFβ1 and TGFβ2 in DMS 53 Cells

shRNA mediated knockdown of TGFβ1 in DMS 53 reduced TGFβ1 secretion by66%. Similarly, shRNA-mediated knockdown of TGFβ2 in DMS 53 reducedTGFβ2 secretion by 53%. The combined knockdown of TGFβ1 and TGFβ2 in DMS53 reduced TGFβ1 secretion by 74% and TGFβ2 secretion by 32% (Table 26)(FIG. 8A).

Knockdown of TGFβ1 and TGFβ2 in NCI-H520 Cells

Knockdown of TGFβ1 in NCI-H520 could not be evaluated because of thelack of detectable TGFβ1 secretion by the parental cell line. Knockdownof TGFβ2 in NCI-H520 reduced TGFβ2 secretion by >99%. The combinedknockdown of TGFβ1 and TGFβ2 in NCI-H520 (ATCC HTB-182) reduced TGFβ2secretion by >99% (Table 26) (FIG. 8B).

Knockdown of TGFβ1 and TGFβ2 in NCI-H2023 Cells

The combined knockdown of TGFβ1 and TGFβ2 in NCI-H2023 reduced TGFβ1secretion below the lower limit of quantification (n=8) resulting in anestimated >90% decrease in TGFβ1 secretion compared to the unmodifiedparental cell line (933±125 μg/10⁶ cells/24h) (n=8). TGFβ1 secretion wassignificantly reduced compared to the unmodified parental cell line(p<0.0002). The combined knockdown of TGFβ1 and TGFβ2 in NCI-H2023reduced TGFβ2 secretion by 65% (118±42 μg/10⁶ cells/24h) (n=8) comparedto the unmodified parental cell line (341±32 μg/10⁶ cells/24h) (n=8).TGFβ2 (p=0.0010) secretion was significantly decreased compared to theunmodified parental cell line (Mann-Whitney U Test) (Table 25) (FIG.9A).

TABLE 25 shRNA mediate reduction of TGFβ1 and TGFβ2 secretion in lungcancer cell lines TGFβ1 (pg/10⁶ cells/24 hours) TGFβ2 (pg/10⁶ cells/24hours) Cell line Parental TGFβ1 KD % Reduction Parental TGFβ2 KD %Reduction NCI-H460 2263 ± 2080 973 ± 551 57 2096 ± 1023 <42 98 NCI-H520<92 <92 NA 3657 ± 3394 <42 >99* DMS 53 504 ± 407 170 ± 128 53 4869 ±5024 3293 ± 4161 32 NCI-H2023 933 ± 125 <92 >90* 341 ± 32  118 ± 42  65NCI-H23 1575 ± 125  644 ± 102 59 506 ± 42  48 ± 9  90 A549 5796 ± 339 914 ± 54  84 772 + 49  42 ± 7  95 NCI-H1703 1736 ± 177  429 ± 133 75 <42<42 NA LK-2 <92 <92 NA 197 ± 34  77 ± 12 61 Parental indicates theunmodified cell line. *Secretion levels are below the lower limit ofquantification for TGFβ1 (92 pg/10⁶ cells/24 hours) or TGFβ2 (42 pg/10⁶cells/24 hours). Lower limit of quantification used to approximate %reduction relative to parental. NA: secretion levels are below the lowerlimit of quantification for both the parental and shRNA modified cellline.

Knockdown of TGFβ1 and TGFβ2 in NCI-H23 Cells

The combined knockdown of TGFβ1 and TGFβ2 in NCI-H23 (ATCC CRL-5800)reduced TGFβ1 secretion by 59% (644±102 μg/10⁶ cells/24h) (n=8) comparedto the unmodified parental cell line (1,575±125 μg/10⁶ cells/24h) (n=8).The combined knockdown of TGFβ1 and TGFβ2 in NCI-H23 (ATCC CRL-5800)reduced TGFβ2 secretion 90% (48±9 μg/10⁶ cells/24h (n=9) compared to theunmodified parental cell line (506±42 μg/10⁶ cells/24h) (n=9). TGFβ1(p=0.0011) and TGFβ2 (p<0.0001) secretion were significantly decreasedcompared to the unmodified parental cell line (Mann-Whitney U Test)(Table 25) (FIG. 9B).

Knockdown of TGFβ1 and TGFβ2 in A549 Cells

The combined knockdown of TGFβ1 and TGFβ2 in A549 reduced TGFβ1secretion by 84% (914±54 μg/10⁶ cells/24h) (n=11) compared to theunmodified parental cell line (5,796±339 μg/10⁶ cells/24h) (n=11). Thecombined knockdown of TGFβ1 and TGFβ2 in A549 reduced TGFβ2 secretion by95% (42±7 μg/10⁶ cells/24h) (n=11) compared to the unmodified parentalcell line (772±49 μg/10⁶ cells/24h) (n=11). Both TGFβ1 (p=0.0128) andTGFβ2 (p=0.0042) secretion were significantly decreased compared to theunmodified parental cell line (Mann-Whitney U Test) (Table 25) (FIG.9C).

Knockdown of TGFβ1 and TGFβ2 in LK-2 Cells

Neither the unmodified parental (n=9) nor the shRNA modified cell lines(n=9) secreted TGFβ1 above the lower limit of quantification of theELISA assay. The combined knockdown of TGFβ1 and TGFβ2 in LK-2 reducedTGFβ2 secretion by 61% (77±12 μg/10⁶ cells/24h) (n=10) compared to theunmodified parental cell line (197±34 μg/10⁶ cells/24h) (n=10). TGFβ2(p=0.0042) secretion were significantly decreased compared to theunmodified parental cell line (Mann-Whitney U Test) (Table 25) (FIG.9D).

Knockdown of TGFβ1 and TGFβ2 in NCI-H1703 Cells

The combined knockdown of TGFβ1 and TGFβ2 in NCI-H1703 reduced TGFβ1secretion by 75% (429±133 μg/10⁶ cells/24h) (n=3) compared to theunmodified parental cell line (1,736±177 μg/10⁶ cells/24h) (n=3). Boththe unmodified parental (n=5) and shRNA modified cell lines (n=5) didnot secret TGFβ2 above the lower limit of quantification of the ELISAassay (Table 25) (FIG. 9E).

Example 6: Downregulation of TGFβ1 and/or TGFβ2 Enhances Cellular ImmuneResponses

Unmodified parental, TGFβ1 KD, TGFβ2 KD, or TGFβ1±β2 KD NCI-H460 cellswere treated with 10 μg/mL MMC for 2 hours and then seeded in 6-wellplate 24 hours prior to the addition of healthy donor PBMCs. PBMCs wereco-cultured with the MMC treated NCI-H460 for 5-6 days in the presenceof IL-2. On day 5 or 6, PBMCs were carefully isolated from theco-culture, counted, and loaded on pre-coated IFNγ ELISpot plates(MabTech). PBMCs were then stimulated with either MMC treated unmodifiedparental NCI-H460 cells or a mixture of 11 peptides comprising known MHCclass I-restricted Survivin epitopes for 36-48 hours. IFNγ SFU weredetected following the manufacturer's instructions, counted (CTL CROScanning Services), and expressed as the number of SFU/10⁶ PBMCs.

Healthy donor (HLA-A*01, HLA-A*02) derived PBMCs sensitized with TGFβ1KD NCI-H460 significantly increases cellular immune responses (1613±187SFU), compared to sensitization with the unmodified parental NCI-H460(507±152 SFU) (p<0.001) (FIG. 10A). Knockdown of both TGFβ1 and TGFβ2also significantly increased IFNγ responses (1823±93 SFU) (p<0.001)compared to unmodified parental NCI-H460. Knockdown of TGFβ2 did notincrease IFNγ production relative to the unmodified parental cell line(390±170 SFU) (p=0.692). The increase in immune responses with knockdownof TGFβ1 and TGFβ2 is likely attributed to the effects of TGFβ1knockdown because TGFβ2 knockdown alone did not enhance immunogenicity.In PBMCs derived from a different donor (HLA-A*01, HLA-A*11) knockdownof TGFβ1 in NCI-H460 significantly increased cellular immune responses(1883±144 SFU), compared to sensitization with the unmodified parentalNCI-H460 (773±236 SFU) (p=0.013) (FIG. 10B). Knockdown of TGFβ2 alone(1317±85 SFU (p>0.999) and of both TGFβ1 and TGFβ2 (1630±62) (p=0.249)also increased IFNγ responses relative to sensitization with unmodifiedparental NCI-H460 cells but did not reach statistical significance.

Survivin (BIRC5) is a well characterized TAA that is overexpressed inmultiple cancer immunotherapy indications. FIG. 10C demonstratessignificantly more robust MHC class I-restricted responses to Survivinin the IFNγ ELISpot assay when donor PBMCs are sensitized with NCI-H460TGFβ2 KD cells (192±120 SFU) compared to unmodified parental NCI-H460cells (28±44) (p=0.005). PBMC sensitization with NCI-H460 TGFβ1 KD(30±64) (p=0.999) or TGFβ1 and TGFβ2 KD (30±38) (p=0.999) did notdemonstrate a significant increase in Survivin specific IFNγ productionin two independent experiments.

The effect of TGFβ1 KD on immunogenicity of this vaccine approach wasfurther characterized in PBMCs isolated from the two healthy donors(HLA-A*24, HLA-A*30) (HLA-A*02, HLA-A*68) in the mixed lymphocyteco-culture reaction (n=3/donor). PBMCs cultured alone, or co-culturedwith NCI-H520 TGFβ1 nonsense control or TGFβ1 KD cells in the presenceof IL-2 for 10 days. PBMCs cultured without tumor cells served as anadditional control. IFNγ secretion was measured in the co-culturesupernatant by ELISA on day 10 (FIG. 11A). IFNγ secretion wassignificantly increased, compared to PBMCs alone (83±86 μg/mL), in thesupernatant of PBMCs co-cultured with NCI-H520 TGFβ1 KD cells (272±259μg/mL) (p=0.046). There was not a significant increase in IFNγ secretionin the supernatant of the NCI-H520 TGFβ1 nonsense KD (86±32 μg/mL)(p=0.512) compared to PBMCs alone.

The impact of TGFβ1 knockdown on the immunogenicity of NCI-H520 wasfurther evaluated in an autologous PBMC DC co-culture assay. DCs,differentiated from monocytes isolated from a healthy donor (HLA-A*24,HLA-A*30), were loaded with cell lysate from NCI-H520 unmodifiedparental cells, TGFβ1 KD, TGFβ2 KD, or TGFβ1±p2 KD cells. AutologousPBMCs were co-cultured with lysate loaded DCs for 5-6 days in thepresence of 20 U/mL of IL-2. On day 5 or 6, PBMCs were carefullyisolated from the co-culture, counted, and 1×10⁵ plated per well onpre-coated IFNγ ELISpot plates (MabTech). PBMCs were then stimulatedwith MMC treated unmodified parental NCI-H520 cells for 36-48 hours. Theresults indicated that there was a trend towards TGFβ1 KD increasingcellular immune responses to NCI-H520 unmodified parental cells (357±181SFU), assayed by IFNγ ELISpot, compared to unmodified parental NCI-H520cells (93±162 SFU) (p=0.181) (FIG. 11B). IFNγ responses to unmodifiedparental NCI-H520 cells induced in autologous PBMCs co-cultured withlysate from NCI-H520 TGFβ2 KD (13±23 SFU) (p=0.897) and TGFβ1 and TGFβ2KD (240±142 SFU) (p=0.603) did not significantly increase IFNγ responsescompared to autologous PBMCs co-cultured with NCI-H520 (ATCC HTB-182)unmodified parental lysate loaded DCs. Despite not reaching statisticalsignificance, cellular immune responses induced by co-culture ofautologous PBMCs with DCs loaded with NCI-H520 TGFβ1 KD and TGFβ1 andTGFβ2 KD were more robust than those with NCI-H520 TGFβ2 KD andunmodified parental lysate.

Example 7: shRNA Downregulation of TGFβ Induces Stronger ImmuneResponses than TGFβ Knockout in Cell Lines

In vitro data suggest that a complete knockout of TGFβ1 and TGFβ2 wasless effective at inducing responses against tumor cells than shRNAknockdown of the two molecules. A representative assay is shown in FIG.12. Normal donor PBMC were cocultured with either TGFβ1/TGFβ2 shRNAmodified or NCI-H460 or TGFβ1/TGFβ2 ZFN knockout NCI-H460 prior toanalysis in an IFNγ ELISpot assay. The data show that the shRNA modifiedcells induced significantly better IFNγ secretion than ZFN-knockoutcells (p=0.0143, unpaired t-test). For this experiment, 5 individualdonors were tested for a total of 24 replicates for the shRNA modifiedcells and 31 replicates for the knockout cells.

Because TGFβ1 is a key player in regulating the epithelial-mesenchymaltransition, complete lack of TGFβ1 induces a less immunogenic phenotypein tumor cells (Miyazono, K et al., Frontiers of Medicine. 2018). Thiswas discernable when compared the ratio of the expression of importantimmune response-related proteins in TGFβ1 TGFβ2 shRNA knockdown inNCI-H460 compared to knockout (FIG. 13). The knockdown cells expressedhigh levels of immunogenic proteins and TAAs compared to the knockoutcells.

Collectively, the data presented in Examples 6 and 7 demonstrate thatreduction of TGFβ1 and/or TGFβ2 can increase cellular immune responsesto unmodified parental tumor cells and antigens in the context of anallogenic whole cell vaccine. Further, these data demonstrate that shRNAmediated knockdown induces more robust immune responses compared toknockout of TGFβ1 and TGFβ2.

Example 8. Immunogenicity of Combinations of Cell Lines with shRNAMediated Downregulated TGFβ1 and/or TGFβ2 Secretion

Immunogenicity of example combinations of cell lines with reduced TGFβ1and/or TGFβ2 secretion were determined by IFNγ ELISpot as described inExample 2 with modifications. Two different responses were evaluated,first for the combinations of cell lines and second for known tumorassociated, tumor-specific, and cancer-testis antigens (collectivelyreferred to as antigens). To assess immune responses generated by thecombinations of cell lines, DCs were loaded at a 1.0:0.33 DC to cellline ratio such that the ratio of DCs to total cell line was 1:1.Specifically, 1.5×10⁶ DCs were cocultured with 5.0×10⁶ cell line 1,5.0e⁵ cell line 2, and 5.0e⁵ cell line 3.

To assess responses to antigens, CD14⁻ PBMCs isolated from co-culturewith mDCs on day 6 were stimulated with antigen specific peptide poolsin the IFNγ ELISpot assay for 24 hours prior to detection of IFNγ SFU.Antigen specific responses are expressed as the number of SFU/10⁶ PBMCsabove that of the controls. Antigen peptide pools were acquired from thecommercial sources as follows: Mage A1 (JPT, PM-MAGEA1), Mage A3 (JPT,PM-MAGEA3), Mage A4 (JPT, PM-MAGEA4), CEACAM (CEA) (JPT, PM-CEA), MUC1(JPT, PM-MUC1), Survivin (thinkpeptides, 7769_001-011), PRAME (MiltenyiBiotec, 130-097-286), WT1 (JPT, PM-WT1), TERT (JPT, PM-TERT), STEAP(PM-STEAP1), and HER2 (JPT, PM-ERB_ECD). Immune responses weredetermined in using cells derived from HLA-A02 (Donors 1-3) and HLA-A11(Donor 4) healthy donors (n=2-3/cell line/donor).

Immunogenicity of the six example combinations of three TGFβ1 and/orTGFβ2 modified cell lines were determined by IFNγ ELISpot (FIG. 14).

Example vaccine cell line Combination 1 was composed of NCI-2023,NCI-H23, and LK-2 TGFβ1 and TGFβ2 modified cell lines. The cell linecombination elicited a total IFNγ response of 5,499±1,016 SFU (n=9/3donors) consisting of 1,800±553 SFU to NCI-2023, 2,069±393 SFU toNCI-H23, and 1,630±102 SFU to LK-2 (FIG. 14A) (Table 26). Examplevaccine cell line Combination 2 was composed of the NCI-H23, DMS 53, andNCI-H1703 TGFβ1 and/or TGFβ2 modified cell lines. This example vaccinecombination elicited a total IFNγ response of 3,604±1,491 SFU (n=9/3donors) consisting of 1,738±529 SFU to NCI-H23, 826±457 SFU to DMS 53,and 1,041±555 SFU to NCI-H1703 (FIG. 14B) (Table 26). Example vaccinecell line Combination 3 was composed of NCI-H2023, DMS 53, and NCI-H1703TGFβ1 and/or TGFβ2 modified cell lines. This example cell linecombination induced a total IFNγ response of 6,065±941 SFU (n=9/3donors) consisting of 2,847±484 SFU to NCI-H2023, 1,820±260 SFU to DMS53, and 1,398±309 SFU to NCI-H1703 (FIG. 14C) (Table 26). Examplevaccine cell line Combination 4 consisted of NCI-H23, DMS 53, and LK-2TGFβ1 and/or TGFβ2 modified cell lines. This example cell linecombination induced a total IFNγ response of 9,612±5,293 SFU (n=12/4donors) consisting of 2,654±1,091 SFU to NCI-H23, 3,017±1,914 SFU to DMS53, and 3,942±2,474 SFU to LK-2. (FIG. 14D) (Table 26). Example vaccinecell line Combination 5 consisted of NCI-H2023, DMS 53, and LK-2 TGFβ1and/or TGFβ2 modified cell lines. This example cell line combinationinduced a total IFNγ response of 6,358±2,278 SFU (n=9/3 donors)consisting of 2,869±1,150 SFU to NCI-H2023, 1,698±568 SFU to DMS 53, and1,791±637 SFU to LK-2 (FIG. 14E) (Table 26). Example vaccine cell lineCombination 6 consisted of NCI-H460, NCI-H520, and A549 TGFβ1 and TGFβ2modified cell lines. This example cell line combination induced a totalIFNγ of 8,407±1,535 SFU (n=12/4 donors) comprising of 2,320±666 SFU toNCI-H460, 2,723±644 SFU to NCI-H520, and 3,005±487 SFU to A549 (FIG.14F) (Table 26).

For some exemplary cell line combinations, IFNγ responses against theindividual unmodified parental cell lines were enhanced when PBMCs wereco-cultured with DCs presenting antigens from three vaccine cell linecombinations relative to PBMCs co-cultured with DCs presenting antigensfrom a single vaccine cell line component (Table 26). The immuneresponses induced by three cell line combinations were more robust thanthe responsed induced by each individual cell line.

TABLE 26 IFNγ responses against cell lines in example combinations oragainst single individual vaccine component cell lines Cell Line ThreeVaccine Cell Single Vaccine Cell Combination 1 Line Combination (SFU)Line Component (SFU) NCI-2023 1,800 ± 553   903 ± 136 NCI-H23 2,069 ±393   1,014 ± 773   LK-2 1,630 ± 102   1,573 ± 935   Cell Line ThreeVaccine Cell Single Vaccine Cell Combination 2 Line Combination (SFU)Line Component (SFU) NCI-H23 1,738 ± 529   1,014 ± 773   DMS 53 826 ±457 227 ± 227 NCI-H1703 1,041 ± 555   724 ± 724 Cell Line Three VaccineCell Single Vaccine Cell Combination 3 Line Combination (SFU) LineComponent (SFU) NCI-H2023 2,847 ± 484   903 ± 136 DMS 53 1,820 ± 260  227 ± 227 NCI-H1703 1,398 ± 309   724 ± 724 Cell Line Three Vaccine CellSingle Vaccine Cell Combination 4 Line Combination (SFU) Line Component(SFU) NCI-H23 2,654 ± 1,091 1,567 ± 788   DMS 53 3,017 ± 1,914 138 ± 85 LK-2 3,942 ± 2,474 1,592 ± 965   Cell Line Three Vaccine Cell SingleVaccine Cell Combination 5 Line Combination (SFU) Line Component (SFU)NCI-H2023 2,869 ± 1,150 903 ± 136 DMS 53 1,698 ± 568   227 ± 227 LK-21,791 ± 637   1,573 ± 935   Cell Line Three Vaccine Cell Single VaccineCell Combination 6 Line Combination (SFU) Line Component (SFU) NCI-H4602,320 ± 666   970 ± 281 NCI-H520 2,723 ± 644   596 ± 336 A549 3,005 ±487   2,677 ± 632  

IFNγ responses to 11 antigens were determined for the example vaccineCombination 4 (NCI-H23, DMS 53, and LK-2 TGFβ1 and/or TGFβ2 modifiedcell lines). Responses against the antigens Mage A1, Mage A3, Mage A4,CEACAM (CEA), MUC1, Survivin, PRAME, WT1, TERT, STEAP, and HER2 wereassessed in 3 HLA-A02 health donors (n=3/donor). Example vaccineCombination 4 induced antigen specific IFNγ responses greater inmagnitude 5,423±427 SFU (FIG. 15A) and breadth (FIG. 15B) compared tothe single vaccine component TGFβ1 and/or TGFβ2 modified cell lines;NCI-H23 (4,1115±2,118 SFU), DMS 53 (3,661±1,982 SFU), and LK-2(2,772±2,936 SFU). Responses to specific antigens are in the orderindicated in the figure legends. The average IFNγ response to eachantigen induced by the single component and combination cell linevaccines are detailed in FIG. 15B.

Example 9: Reduction of HLA-E Expression Improves Cellular ImmuneResponses

HLA-E belongs to the HLA class I heavy chain paralogues. Human tumorcell surface expression of HLA-E can inhibit the anti-tumor functions ofNK, DC, and CD8 T cells through binding to the NKG2A receptor on theseimmune cell subsets.

Reduction of HLA-E Expression in the RERF-LC-Ad1 Cell Line (JCRB1020)

The human adenocarcinoma cell line RERF-LC-Ad1 was electroporated with azinc finger nuclease (ZFN) pair specific for HLA-E targeting thefollowing genomic DNA sequence:TACTCCTCTCGGAGGCCCTGgcccttACCCAGACCTGGGCGGGT (SEQ ID NO: 33).Full-allelic knockout cells were identified by flow cytometry afterstaining with PE-conjugated anti-human HLA-E (BioLegend, clone 3D12)then FACS sorted. Cells were expanded after sorting and percent knockoutdetermined. The MFI of the unstained control of the HLA-E KO orunmodified parental cell was subtracted from the MFI of the HLA-E KO orunmodified parental cells stained with PE-conjugated anti-human HLA-E(BioLegend, clone 3D12). Gene editing of HLA-E by ZFN resulted ingreater than 99% HLA-E negative cells after FACS sorting (FIG. 16A).Knockout percentage is expressed as: (RERF-LC-Ad1 HLA-E KO MFI/ParentalMFI)×100.

Reduction of HLA-E Expression Improves Immune Responses

IFNγ ELISpot was completed as described in Example 8 with onemodification. In this experiment iDC were loaded with only one cellline, RERF-LC-Ad1 parental or HLA-E KO cell lines. Here, 1.5×10⁶ DCswere loaded with 1.5×10⁶ RERF-LC-Ad1 parental or HLA-E KO cells. IFNγresponses were 1.8-fold higher when autologous PBMCs were co-culturedwith DCs loaded with HLA-E negative cells (5085±1157 SFU) relative toDCs loaded with the unmodified parental HLA-E positive cells (2810±491SFU). Student's test, p=0.012. n=12, 3 HLA-A diverse donors (FIG. 16B).

Example 10: Reduction of Cytotoxic T-Lymphocyte-Associated Protein 4(CTLA-4) Expression Increases Cellular Immune Responses

CTLA-4 (CD152) functions as an immune checkpoint and is constitutivelyexpressed on some tumor cells. CTLA-4 binding to CD80 or CD86 on thesurface of DCs can negatively regulate DC maturation and inhibitproliferation and effector function of T cells.

Reduction of CTLA-4 Expression in Human Squamous Cell Line

The NCI-H520 cell line was transfected with siRNA targeting CTLA-4(Dharmacon, L-016267-00-0050). Cells were seeded at 6×10⁵ in each wellof a six well plate in antibiotic-free media and incubated at 37° C. in5% CO₂. Following DharmaFect siRNA transfection protocol, each well wastransfected with a 25 nM final concentration of CTLA-4 siRNA using 4 uLof DharmaFECT 1 Transfection Reagent (Dharmacon, T-20001-01) per well.Reduction of CTLA-4 expression on live cells was determined by flowcytometry 72 hours after siRNA transfection prior to use in the IFNγELISpot assay. Specifically, NCI-H520 cells were stained with LIVE/DEAD™Aqua (Invitrogen, L34965) and human α-CTLA4-APC (BioLegend, cloneL3D10). siRNA reduced NCI-H520 cell surface expression of CTLA-4 (3.59%)2.1-fold compared to unmodified parental NCI-H520 (7.59%) (FIG. 17A).

Reduction of CTLA-4 Expression in the NCI-H520 (ATCC HTB-182) Cell LineIncreases Cellular Immune Responses

The impact of reducing cell surface expression of CTLA-4 on cellularimmune responses was evaluated in the IFNγ ELISpot assay using cellsderived from an HLA-A 02:01 donor. The ELISpot was initiated 72 hoursafter siRNA transfection and carried out as described in Example 9.Reduction of CTLA-4 expression in NCI-H520 was associated with a1.6-fold increase in IFNγ responses (2,770±180 SFU) (n=2) compared tothe unmodified parental cell line (1,730±210 SFU) (n=2) (FIG. 17B).

Example 11. Reduction of CD276 Expression in the A549 Cell Line EnhancesCellular Immune Responses

CD276 (B7-H3) is an immune checkpoint member of the B7 and CD28families. Over expression of CD276 in human solid cancers can induce animmunosuppressive phenotype and preferentially down-regulatesTh1-mediated immune responses.

Reduction of CD276 expression in A549 was completed using theCRISPR-Cas9 system with guide RNA specific for TGCCCACCAGTGCCACCACT (SEQID NO: 117)(Synthego). The initial heterogenous population contained 71%A549 cells where CD276 expression was reduced. The heterogenouspopulation was surface stained with BB700-conjugated α-human CD276 (BDBiosciences, clone 7-517) and full allelic knockout cells enriched bycell sorting (BioRad S3e Cell Sorter). The reduction of CD276 wasconfirmed by extracellular staining of the sort enriched A540 CD276 KOcells and parental A549 cells with PE α-human CD276 (BioLegend, cloneDCN.70). Unstained and isotype control PE α-mouse IgG1 (BioLegend, cloneMOPC-21) stained A549 CD276 KO cells served as controls. Cas9-mediatedgene editing of CD276 resulted in >99% reduction of CD276 compared tocontrols (FIG. 18A).

In a representative experiment, iDCs were loaded A549 parental cells orA549 CD276 KO cells and co-cultured with autologous CD14-PBMCs for 6days prior to stimulation with autologous DCs loaded with cell lysatefrom wild type A549. Cells were then assayed for IFNγ secretion againstwild type A549 cells in an ELISpot assay. These data show that CD276 KOcells are better stimulators than the wild type cells (p=0.017; unpairedt test) (FIG. 18B).

Example 12: Reduction of CD47 Expression and TGFβ1 and/or TGFβ2Secretion

Methods for shRNA downregulation of TGFβ1 and TGFβ1 and determine levelsof secreted TGFβ1 and TGFβ2 are described in Example 5.

Reduction of CD47 Expression in Human Lung Cancer Lines with shRNADownregulated TGFβ1 and or TGFβ2

The A549, NCI-H460, NCI-H2023, NCI-H23, NCI-H520, LK-2, and NCI-H1703that were modified to decrease secretion of TGFβ1 and/or TGFβ2 werefurther modified to reduce expression of CD47 as described in Example 2and additional methods described here. Following ZFN-mediated knockoutof CD47, the cell lines were surface stained with FITC-conjugated α-CD47(BD Biosciences, clone B6H12) and full allelic knockout cells enrichedby cell sorting (BioRad S3e Cell Sorter). The cells were collected usinga purity sorting strategy to ensure the collection of only CD47 negativecells. The sorted cells were plated in an appropriately sized vesselbased on cell number, grown and expanded. After cell enrichment for fullallelic knockouts, the TGFβ1 and/or TGFβ2 KD CD47 KO cells were passaged2-5 times and CD47 knockout percentage determined by flow cytometry(BV421-conjugated human αCD47, BD Biosciences, clone B6H12). The MFI ofthe unstained controls for the modified or unmodified parental cellswere subtracted from the MFI of the modified or unmodified parentalcells stained with BV421-conjugated human α-CD47. CD47 knockoutpercentage is expressed as: (1-(TGFβ1/TGFβ2 KD CD47 KO MFI/ParentalMFI))×100).

Gene editing of CD47 by ZFN resulted in greater than 99% CD47 negativecells after FACS sorting in the cell lines (Table 27) while maintainingreduced secretion of TGFβ1 and/or TGFβ2 (Table 28). The downregulationof TGFβ1 and/or TGFβ2 with reduction of CD47 expression is shown asfollows: NCI-H2023 in FIG. 19, NCI-H23 in FIG. 20, A549 in FIG. 21,NCI-H460 in FIG. 22, NCI-H1703 in FIG. 23, LK-2 in FIG. 24, DMS 53 inFIG. 25, and NCI-H520 in FIG. 26.

TABLE 27 CD47 KO in TGFβ1 and/or TGFβ2 KD cell lines Parental ModifiedCell line CD47 MFI CD47 MFI % Reduction CD47 NCI-H2023 244,674 0 100.0NCI-H23 252,210 1745 99.3 A549 96,845 29 99.9 NCI-H460 134,473 343 99.7NCI-H1703 202,482 1069 99.5 LK-2 92,360 0 100.0 DMS 53 46,399 389 99.2NCI-H520 158,037 145 99.9 MFI reported with unstained controlssubtracted. Parental indicates the unmodified cell line.

TABLE 28 TGFβ1 and TGFβ2 secretion in TGFβ1 and/or TGFβ2 KD cell linesCD47 KO cell lines TGFβ1 (pg/10⁶ cells/24 hours) TGFβ2 (pg/10⁶ cells/24hours) Cell line Parental TGFβ1 KD % Reduction Parental TGFβ2 KD %Reduction NCI-H2023 1262 ± 163 <92 >93* 393 ± 168 168 ± 57  57 NCI-H231993 ± 540 590 ± 136 70 679 ± 211 <42 >94* A549 5962 ± 636 952 ± 77  84718 ± 82  45 ± 12 94 NCI-H460 1758 ± 75  227 ± 45  87 2564 ± 200  559 ±147 57 NCI-H1703 1700 ± 300 565 ± 91  67 <42 <42 NA LK-2 <92 <92 NA 111± 41  58 ± 13 48 DMS 53 Not completed 2458 ± 675  1409 ± 313  43NCI-H520 <92 <92 NA 3278 ± 837  151 ± 13  95 Parental indicates theunmodified cell line. *Secretion levels are below the lower limit ofquantification for TGFβ1 (92 pg/10⁶cells/24 hours) or TGFβ2 (42pg/10⁶cells/24 hours). Lower limit of quantification used to approximate% reduction relative to parental. NA: secretion levels are below thelower limit of quantification for both the parental and shRNA modifiedcell line.

Example 13: Reduction of CD276 Expression and TGFβ1 and/or TGFβ2Secretion Increases Cellular Immune Responses

The human tumor cell lines NCI-H460, NCI-H520, DMS 53, A549, NCI-H2023,NCI-H23, LK-2 and NCI-H1703, in which TGFβ1 and/or TGFβ2 secretion wasreduced by shRNA in Example 5 were electroporated with a zinc fingernuclease (ZFN) pair specific for CD276 targeting the genomic DNAsequence: GGCAGCCCTGGCATGggtgtgCATGTGGGTGCAGCC. (SEQ ID NO: 26).Following ZFN-mediated knockout of CD276 in the TGFβ1 and/or TGFβ2 KDlines, the cell lines were surface stained with BB700-conjugated α-humanCD276 (BD Biosciences, clone 7-517) and full allelic knockout cellsenriched by cell sorting (BioRad S3e Cell Sorter). The cells werecollected using a purity sorting strategy to ensure the collection ofonly CD276 negative cells. The sorted cells were plated in anappropriately sized vessel based on cell number, grown and expanded.After cell enrichment for full allelic knockouts, the TGFβ1 and/or TGFβ2KD CD276 KO cells were passaged 2-5 times and CD276 knockout percentageby flow cytometry (BV421-conjugated human α-CD276, BD Biosciences, clone7-517). The MFI of the unstained controls for modified cells orunmodified parental cells were subtracted from the MFI of the modifiedcells or unmodified parental cells stained with BV421-conjugated humanα-CD276. Percent reduction is expressed as: (1-(TGFβ1/β2 KD CD276 KOMFI/Parental MFI))×100).

Gene editing of CD276 by ZFN resulted in greater than 99% CD276 negativecells (Table 29) in the cell lines with reduced secretion of TGFβ1and/or TGFβ2 (Table 31). The downregulation of TGFβ1 and/or TGFβ2 withreduction of CD276 expression is shown as follows: NCI-H2023 in FIG. 27,NCI-H23 in FIG. 28, A549 in FIG. 29, NCI-H460 in FIG. 30, NCI-H1703 inFIG. 31, LK-2 in FIG. 32, DMS 53 in FIG. 33, and NCI-H520 in FIG. 34.

TABLE 29 CD276 knockout in cell lines with reduced TGFβ1 and/or TGFβ2secretion. Parental Modified Cell line CD276 MFI CD276 MFI % ReductionCD276 NCI-H2023 262,460 680 99.7 NCI-H23 74,176 648 99.1 A549 141,009688 99.5 NCI-H460 366,565 838 99.8 NCI-H1703 262,386 417 99.9 LK-2385,535 867 99.8 DMS 53 304,637 972 99.7 NCI-H520 341,202 212 99.9 MFIreported with unstained controls subtracted. Parental indicates theunmodified cell line.

TABLE 30 TGFβ1 and TGFβ2 secretion in TGFβ1 and/or TGFβ2 KD CD276 KOcell lines. TGFβ1 (pg/10⁶ cells/24 hours) TGFβ2 (pg/10⁶ cells/24 hours)Cell line Parental TGFβ1 KD % Reduction Parental TGFβ2 KD % ReductionNCI-H2023 1090 ± 279 97 ± 23 91 347 ± 57  153 ± 93  56 NCI-H23 1683 ±111 706 ± 180 58 523 ± 37  55 ± 18 89 A549 6443 ± 406 770 ± 29  88 757 ±125 61 ± 8  92 NCI-H460 1415 ± 282 390 ± 14  72 2100 ± 542  680 ± 166 68NCI-H1703 1682 ± 155 434 ± 53  74 <42 <42 NA LK-2 <92 <92 NA 140 ± 64 76 ± 16 46 DMS 53 Not completed 4053 ± 2548 2329 ± 1175 52 NCI-H520 <92<92 NA 4045 ± 525  59 ± 34 99 Parental indicates the unmodified cellline NA: secretion levels are below the lower limit of quantificationfor both the parental and shRNA modified cell line.

TGFβ1 and TGFβ2 KD and CD276 KO increases cellular immune responses

IFNγ ELISpot was carried out as described in Example 9. Cells derivedfrom HLA-A02 and HLA-A03 healthy donors were used to evaluate ifreduction of TGFβ1 and TGFβ2 secretion and CD276 expression couldimprove immune responses compared to the unmodified parental cell lines.In the NCI-H460 cell line, modification of TGFβ1, TGFβ2, and CD276increased IFNγ responses 2.3-fold (569±87 SFU) (n=11) relative to theunmodified parental cell line (250±63 SFU) (n=11) (p=0.0078,Mann-Whitney U Test) (FIG. 35A). In the A549 cell line, modification ofTGFβ1, TGFβ2 and CD276 increased IFNγ responses 22.2-fold (83±29 SFU)(n=11) relative to the unmodified parental cell line (1,848±569 SFU)(n=11) (p=0.0091, Mann-Whitney U Test) (FIG. 35B).

Example 14: Reduction of CD276 and CD47 Expression and TGFβ1 and TGFβ2Secretion Increases Cellular Immune Responses

The A549 cell line was modified to reduce TGFβ1 and TGFβ2 secretionusing shRNA and reduce expression of CD47 and CD276. Methods used tosecretion and determine levels of TGFβ1 and TGFβ2 are described inExample 5. Methods employed to reduce expression of CD47 and CD276 anddetermine expression levels are described in Example 12 and Example 13,respectively. IFNγ ELISpot was completed as described in Example 9.

Characterization of A549 Cells with Reduced Expression of CD276 and CD47and TGFβ1 and TGFβ2 Secretion

CD47 expression was reduced 99.9% on the modified cell line (136 MFI)relative to the unmodified parental cell line (104,442 MFI) (FIG. 36A)(Table 31). CD276 expression was reduced 100% on the modified cell line(0 MFI) relative to the unmodified parental cell line (53,196 MFI) (FIG.36B) (Table 31). TGFβ1 secretion was by the modified cell line (2027±31μg/10⁶ cells/24 hours) (n=2) was reduced 78% compared to the unmodifiedparental cell line (9093±175 μg/10⁶ cells/24 hours) (n=2) (FIG. 36C).TGFβ2 secretion by the modified cell line was below the lower limit ofquantification of the ELISA assay (n=2), resulting in a 100% reductionin secretion levels relative to the unmodified parental cell line(607±76 μg/10⁶ cells/24 hours) (n=2) (FIG. 36D).

Reduction of CD276 and CD47 Expression and TGFβ1 and TGFβ2 SecretionIncreases Cellular Immune Responses

Cells derived from HLA-A02 (FIG. 37A), HLA-A03 (FIG. 37A), and HLA-A24(FIG. 37B) healthy donors were utilized in the IFNγ ELISpot assay todetermine if modification of TGFβ1 and TGFβ2, CD276, and CD47 in theA549 cell line enhanced immune responses relative to the unmodifiedparental cell line. IFNγ ELISpot was completed as described in Examples9. The modified cell line increased IFNγ responses 26.8-fold (83±29 SFU)(n=11) relative to the unmodified parental cell line (2,233±493 SFU)(n=11) (p=0.0091, Mann-Whitney U test) (FIG. 37A). Responses against 10antigens were assessed for the unmodified parental, TGFβ1 TGFβ2 KDCD47KO, TGFβ1 TGFβ2 KD CD276 KO, and TGFβ1 TGFβ2 KD CD276 CD47KO A549modified cell lines. Relative to the total TAA response induced by theunmodified parental cell line (15,140 SFU) (n=3), reduction of TGFβ1,TGFβ2, and CD47 increased the total antigen specific response 1.7-fold(25,813 SFU) (n=3), reduction of TGFβ1, TGFβ2, and CD276 increased thetotal antigen specific response 2.0-fold (30,640 SFU) (n=3), andreduction of TGFβ1, TGFβ2, CD47 and CD276 increased the total TAAresponse 2.0-fold (29,993 SFU) (n=3) (FIG. 37B). Responses to specificantigens are in the order indicated in the figure legends. The datasuggests that both reduction of CD47 and/or CD276 concurrently withreduction in TGFβ1 and TGFβ2 secretion can promote increasedTAA-specific IFNγ production.

TABLE 31 Knockout of CD47 or CD276 in TGFβ1 and TGFβ2 KD cell linesmodified to secrete GM-CSF, express membrane bound CD40L, and secreteIL-12. Cell line Parental MFI CD47 MFI % Reduction A549 100,228 33 99.9NCI-H460 140,990 6 >99.9   Cell line Parental MFI CD276 MFI % ReductionA549 30,636 326 98.9 NCI-H460 82,858 1,467 98.2 MFI reported withunstained controls subtracted. Parental indicates the unmodified cellline.

Example 15: Expression of Membrane Bound CD154 (Membrane Bound CD40Ligand) Enhances Cellular Immune Responses

CD40 Ligand (CD40L) is transiently expressed on T cells and othernon-immune cells under inflammatory condition and binds to thecostimulatory molecule CD40 on B cells and professionalantigen-presenting cells. The binding of CD40L to CD40 upregulatesmultiple facets of adaptive cellular and humoral immunity.

Expression of Membrane Bound CD40L in the A549 Cell Line

The cell line A549 cell line was transduced with lentiviral particlesexpressing a CD40L sequence modified to reduce cleavage by ADAM17 and,thereby, promote membrane bound CD40L expression. Parental, unmodifiedcell lines served as controls. After antibiotic selection in 200 μg/mLto enrich for cells stable expressing CD40L, cells were analyzed forCD40L expression on the cell surface using flow cytometry andsolubilized CD40L detected by ELISA. The sequence of membrane boundCD40L used in this example is shown in SEQ ID NO: 1.

To determine the level of membrane bound CD40L expression, unmodifiedparental and modified cells were stained with PE-conjugated humanα-CD40L (BD Biosciences, clone TRAP1). There was a 25.5-fold increase inthe expression of CD40L on the cell surface (43,466 MFI) compared to theunmodified parental A549 cell line (1702 MFI) (FIG. 38A).

Solubilized CD40L was quantified by ELISA. CD40L-transduced andunmodified parental cells were plated at 8.33×10⁴ cells/well in a24-well plated in regular growth medium (RPMI containing 10% FBS).Twenty-four hours after plating, adherent cells were thoroughly washedto remove FBS and culture was continued in RPMI+5% CTS. Forty-eighthours after media replacement, the cell culture supernatant washarvested, and stored at −70° C. until the assays were completedaccording to the manufacturers instructions (BioLegend, DCDL40). Thelower limit of quantification of human CD40L is 62.5 μg/mL, or 0.375ng/10⁶ cells/24 hours. Overexpression of CD40L resulted in 2.93 ng/10⁶cells/24 hours of sCD40L (FIG. 38B).

The effect of A549 CD40L expression on DC maturation was characterizedby flow cytometry. iDCs and A549 unmodified parental cells, unmodifiedparental cells with exogenous sCD40L (1 μg/mL) (PeproTech,#AF31002100UG), or A549 cells overexpressing membrane-bound CD40L wereco-cultured at a 1:1 ratio in 96-well low-adherence U bottom plates.Following the 24 hours incubation, the co-cultures were surface stainedwith LIVE/DEAD Aqua (Molecular Probes, #L23105), αCD45-PE-Cy7 (BDBiosciences, clone H130), and αCD11c-BV605 (BD Biosciences, cloneB-Iy6), and αCD83-APC (BD Biosciences, clone HB15e). Flow cytometry datawas analyzed using FlowJo (FlowJo LLC). Increased DC maturation wasdefined as an increase in the % live, CD45⁺CD11c⁺CD83⁺ DCs. DCmaturation was evaluated for 7 HLA diverse healthy donors.

A549 expression of CD40L significantly increased the % of live,CD45⁺CD11c⁺CD83⁺ DCs 3.9-fold (40±5) relative to the unmodified parentalcell line (10±3) (p<0.001, Holm-Sidak's multiple comparisons test)(n=7). Exogenous sCD40L did not significantly increase the % of live,CD45⁺CD11c⁺CD83⁺ DCs (16±3) (p=0.4402, Holm-Sidak's multiple comparisonstest) (n=7) (FIG. 38C).

Expression of Membrane Bound CD40L Enhances Cellular Immune Responses

The effect of overexpression of CD40L on induction of cellular immuneresponses was evaluated by IFNγ ELISpot assay as described in Example 9.iDCs loaded were loaded with A549 cells, A549 cells with 1 μg/mLexogenous sCD40L, or A549 cells overexpressing CD40L. Expression ofCD40L by A549 cells increased IFNγ responses 87-fold (1,305±438 SFU)compared to the unmodified parental cell line (15±15 SFU) (p=0.0198,Holm-Sidak's multiple comparisons test) (n=4). Inclusion of exogenoussCD40L in the co-culture did not significantly increase IFNγ responses(255±103 SFU) relative to the unmodified parental cell line (p=0.5303,Holm-Sidak's multiple comparisons test) (n=4). IFNγ responses elicitedby overexpression of CD40L on A549 cells were significantly greater thanthe responses detected with the addition of exogenous sCD40L (p=0.0375,Holm-Sidak's multiple comparisons test) (n=4) (FIG. 38D).

Example 16: Expression of GM-CSF Enhances Cellular Immune Responses

Unmodified parental NCI-H460 cells were transfected with either emptylentiviral vector (control) or a lentiviral vector designed tooverexpress GM-CSF (SEQ ID NO: 6). The control and GM-CSF overexpressing cell line were grown in the presence of Puromycin (2 μg/mL)prior to use in the IFNγ ELISpot assay. IFNγ ELISpot was performed asdescribed in Example 6. FIG. 39 demonstrates that sensitization ofhealthy donor (HLA-A*01, HLA-A*02) derived PBMCs with GM-CSFoverexpressing NCI-H460 cells significantly increases cellular immuneresponses to unmodified parental NCI-H460 cells (2600±207 SFU) whencompared to sensitization with the Control NCI-H460 cells (1163±183 SFU)(p=0.002).

Example 17: Expression of Interleukin-12 (IL-12) Enhances CellularImmune Responses

IL-12 is a proinflammatory cytokine that promotes DCs and LCs to prime Tcells towards an effector phenotype. IL-12 can also act directly on DCsto reverse or prevent the induction of immune tolerance.

The A549 cells were transduced with lentiviral particles expressing boththe p40 and p35 chains of IL-12 to form the functional IL-12 p70cytokine protein. The p40 and p35 sequences are separated by a P2Acleavage sequence. The sequence of IL-12 used in this example is shownin SEQ ID NO: 9. Unmodified parental, unmodified cell lines served ascontrols. After antibiotic selection in 600 μg/mL zeocin to enrich forcells stably expressing IL-12 immune responses generated by the parentaland IL-12 modified cell lines were determined as described in Example 9.There was a 16-fold increase in IFNγ SFU with the expression of IL-12(873±199 SFU) (n=3) compared to IFNγ responses induced by the unmodifiedparental cells (53±53 SFU) (p=0.0163, Mann-Whitney U test) (n=3) (FIG.40).

Example 18: Expression of Glucocorticoid-Induced TNFR Family RelatedGene (GITR) Enhances Cellular Immune Responses

GITR is surface receptor molecule involved in inhibiting the suppressiveactivity of T-regulatory cells (Tregs) and extending the survival ofT-effector cells. Binding of GITR to its ligand, GITR, on APCs triggerssignaling which co-stimulates both CD8⁺ and CD4⁺ effector T cells,leading to enhanced T cell expansion and effector function, whilesuppressing the activity of Tregs.

Expression of GITR

A codon optimized sequence was generated based on the native, membranebound variant of GITR (NP_004186) as and cloned in to the BamHI and XhoIrestriction endonuclease site of pVAX1 (Invitrogen, #V26020)(GenScript). The sequence of GITR used in this example is shown in SEQID NO: 4. For transfections of cells using pVAX1 encoding GITR, A549(5.38×10⁶ cells), NCI-H460 (1.79×10⁷ cells), LK-2 (2.39×10⁷ cells) orNCI-H520 (1.02×10⁷ cells) were plated into T175 flasks using 45 mL ofcomplete culture media 18-24 hours prior to transfection and maintainedat 37° C./5% CO₂. Plasmid DNA transfections were performed using theLipofectamine transfection reagent (Invitrogen, #2075084) according tothe manufacturer's instructions. Cells were incubated at 37° C. and 5%CO₂ for 72 hours prior to assessment of GITR expression by flowcytometry.

To determine cell surface expression of GITR, transfected cells andunmodified parental controls were surfaced stained with BV421-conjugatedmouse anti-human GITR antibody (BD Biosciences, clone V27-580). Flowcytometry data was acquired on a BD LSRFortessa and analyzed usingFlowJo software. Minimal expression of GITR was detected onuntransfected unmodified parental cell lines (n=3 for each cell line)(FIG. 41). GITR was expressed on 17.7±0.1% of transfected NCI-H520 cells(n=3) (FIG. 41A), 29.3±3.3% of transfected LK-2 cells (n=3) (FIG. 41B),7.7±0.2% of transfected A549 cells (n=3) (FIG. 41C), and 14.1±0.9% oftransfected NCI-H460 cells (n=3) (FIG. 41D).

Expression of GITR Enhances Cellular Immune Responses

The effect of expression of GITR on cellular immunogenicity wasevaluated by IFNγ ELISpot as described in Example 9 using cells derivedfrom two HLA-A02 donors and one HLA-A24 healthy donor (n=3/donor).Expression of GITR by the A549 cell line significantly increased IFNγproduction 7.4-fold (947±217 SFU) (n=9) compared to the unmodifiedparental A549 cell line (128±38 SFU) (n=9) (p=0.0003, Mann-Whitney Utest) (FIG. 42A). There was a trend towards increased IFNγ productionwith expression of GITR in the LK-2 cell line (1,053±449 SFU) (n=9)compared the unmodified parental cell line (773±255 SFU) (n=9) (FIG.42B). There was a trend towards increased immunogenicity with GITRexpression in the NCI-H520 cell line (2,953±504 SFU) (n=3) compared tothe unmodified parental, unmodified cells (1,953±385 SFU) (n=3) (FIG.42C). There was also a trend towards increased immunogenicity with GITRexpression in the NCI-H460 (4,940±557 SFU) cell line compared to theunmodified parental cells (3,400±181 SFU) (n=3) (FIG. 42D).

Example 19: Expression of Interleukin-15 (IL-15) Enhances CellularImmune Responses

IL-15 is a member of the four α-helix bundle family of cytokines and isproduced by a wide range of cells including DCs and is essential for thedifferentiation of CD8⁺ memory T□cells. Two isoforms of IL-15 arenatively expressed that encode two different N-terminal signal peptides.These signal peptides function to decrease or inhibit secretion of theIL-15 protein from tumor cells. A codon optimized sequence of IL-15 wasgenerated where the native IL-15 long signal peptide region was replacedwith IL-2 signal peptide to promote secretion of the IL-15 protein(GenScript). The codon optimized sequence was cloned into the BamHI andXhoI restriction sites of pVAX1. The sequence of IL-15 used in thisexample is shown in SEQ ID NO: 11.

Quantification of IL-15 Secretion

Transfections of the IL-15 encoding plasmid were completed as describedin Example 18. Supernatants were assayed for the presence of secretedIL-15 by ELISA using the Human IL-15 Quantikine ELISA Kit (R&D Systems,D1500) and following the manufacturers instructions. The lower limit ofquantification of the IL-15 ELISA is 3.98 μg/mL, or 0.0239 ng/10⁶cells/24 hours. The NCI-H520, LK-2, NCI-H460, and A549 cell linesexpressed 9.04, 5.99, 59.43, and 34.74 ng/10⁶ cells/24 hours of IL-15,respectively (FIG. 43A).

IL-15 Enhances Cellular Immune Responses

IFNγ ELISpot to evaluate the effect of IL-15 on cellular immuneresponses was completed as described in Example 9. The effect of IL-15secretion by the NCI-H460 cell line on cellular immune responses wasevaluated using immune cells derived from an HLA-A02 healthy donor(n=3). There was a trend towards increased IFNγ production with IL-15overexpression (5,593±474 SFU) relative to the unmodified parentalNCI-H460 cell line (4,360±806 SFU) (FIG. 43B).

Example 20: Expression of Interleukin-23 (IL-23) Enhances CellularImmune Responses

IL-23 is a binary complex of a four-helix bundle cytokine (p19) and asoluble class I cytokine receptor p40. IL-23 acts as a proinflammatorycytokine that enhances DC maturation and suppresses DC activation ofnaive T cell-derived Tregs.

Expression of IL-23

Human codon optimized IL-23 p19 and p40 sequences were generated andcloned into the BamHI and XhoI restriction sites of pVAX1 (GenScript).The p19 and p40 sequences were separated by a flexible linker GS3linker. The sequence of IL-23 used in this example is shown in SEQ IDNO: 13. Transfections were completed as described in Example 18.

Supernatants were assayed for the presence of functional (p19 and p40dimers) secreted IL-23 using the Human IL-23 Quantikine ELISA Kit (R&DSystems, D2300B) according to the manufacturer's instructions. The lowerlimit of quantification of the IL-23 ELISA is 39.1 μg/mL, or 0.235ng/10⁶ cells/24 hours. The LK-2 and A549 cell lines expressed 1,559 and1,929 ng/10⁶ cells/24 hours of IL-23, respectively (FIG. 44A).

Secretion of IL-23 Increases Cellular Immune Responses

IFNγ ELISpot to evaluate the effect of IL-23 on cellular immuneresponses was completed as described in Example 9. The effect of IL-15secretion by the A549 (ATCC CCL-185) cell line on cellular immuneresponses was evaluated using immune cells derived from an HLA-A02healthy donor. There was a significant 3.9-fold increase in IFNγproduction with IL-23 overexpression (2,247±580 SFU) relative to theunmodified parental A549 (ATCC CCL-185) cell line (573±401 SFU) (FIG.44B) (p=0.0284, Student's T-test) (n=3).

Example 21: Expression of X-C Motif Chemokine Ligand 1 (XCL1)

The cytokine XCL1, also known as Lymphotactin, binds to the chemokinereceptor XCR1, which is selectively expressed on antigencross-presenting DCs. Expression of XCL1 has the potential to functionas an adjuvant for intradermal vaccine administration.

Expression of XCL1

A human codon optimized sequence was generated encoding human XCL1(GenScript) and cloned into the BamHI and XhoI restriction sites of thepVAX1 plasmid. Transient expression and secretion of XCL1 wascharacterized by ELISA. The sequence of XCL1 used in this example isshown in SEQ ID NO: 15.

Quantification of XCL1 Secretion

NCI-H460 and A549 cells were transfected with pVAX1 encoding codonoptimized XCL1 as described in Example 18. Twenty-four hours aftertransfection, supernatants were removed from the cells and assayed forthe presence of secreted XCL1 by ELISA. Supernatants were assayed forXCL1 secretion according to the manufacturer's instructions (R&DSystems, #DXCL10). The NCI-H460 and A549 cell lines transientlyexpressed 418 and 144 and ng/10⁶ cells/24 hours of XCL1, respectively(FIG. 45).

Example 22: Expression of Mesothelin (MSLN)

MSLN is expressed on the surface of many lung adenocarcinomas andexpression is correlated with poor prognosis. MSLN is an attractive TAAtargeted because antigen specific immune responses to MSLN can predictthe survival of patients with brain metastasis resulting from severaldifferent primary tumors including ovarian, lung and melanoma. A smallsubset of lung cancer cell lines express MSLN despite expression of MSLNin many patient tumors. In Example 22, the expression of MSLN wasgenetically introduced in exemplary vaccine cell lines that do notnatively express MSLN to broaden the coverage TAAs potentially importantto patients with NSCLC.

Expression of MSLN

A codon optimized human MSLN sequence was generated in which the ADAM17cleavage site replaced with a flexible linker to promote retention ofMSLN in the cell membrane (GenScript). The codon optimized sequence wascloned into the BamHI and XhoI restriction sites of pVAX1. The sequenceof MSLN used in this example is SEQ ID NO: 17.

Quantification of MSLN Expression

Transfections of the MSLN encoding plasmid were completed as describedin Example 18. To determine cell surface expression of MSLN, transfectedcells and unmodified parental controls were surfaced stained withPE-conjugated rat anti-human MSLN antibody (R&D Systems, FAB32652P).Flow cytometry data was acquired on a BD LSRFortessa and analyzed usingFlowJo software. Minimal expression of MSLN was detected onuntransfected, unmodified parental cell lines (n=3/cell line) (FIG. 46).MSLN was expressed on 34.7±2.2% of transfected NCI-H520 cells (n=3)(FIG. 46A), 41.4±0.7% of transfected LK-2 cells (n=3) (FIG. 46B),34.6±0.7% of transfected A549 cells (n=3) (FIG. 46C), and 48.5±1.3% oftransfected NCI-H460 cells (n=3) (FIG. 46D).

MSLN-Specific IFNγ Responses

Immune responses to the overexpressed MSLN antigen were characterized byIFNγ ELISpot. To detect MSLN-specific responses in this assay, peptides15 amino acids in length, overlapping by 11 amino acids, were generatedto cover the native protein MSLN protein and used to stimulate PBMCs asdescribed in Example 8. IFNγ responses to the overexpressed MSLN protein(240 SFU) in LK-2 (FIG. 46E).

Example 23: Expression of Kita-Kyushu Lung Cancer Antigen 1 (CT83)

CT83 is expressed by 40% non-small-cell lung cancer tissues and by 31%Stage 1 NSCLC. CT83 is highly expressed in lung tumors compared tonormal tissue. Expression of CT83 is also typically associated with poorprognosis. In Example 23, the expression of CT83 was geneticallyintroduced in exemplary vaccine cell lines that do not natively expressCT83 to broaden the coverage TAAs potentially relevant to some NSCLCpatients.

Expression of CT83

A codon optimized sequence of human CT83 was generated and cloned inframe with codon optimized MSLN (Example 17). SEQ ID NO: 21 was used.The MSLN and CT83 coding sequences were separated by a P2A cleavage siteand cloned into the BamHI and XhoI restriction sites of pVAX1.

Characterization of CT83 Expression

Expression of CT83 by pVAX1-MSLN-CT83 was determined by western blot.Transfections were completed as described in described in Example 18.Transfected cells were lysed by the addition of 100 μL 1×NuPAGE® LDSSample Buffer (Invitrogen, #NP0007) and incubated for 5 minutes at roomtemperature. The cell lysate was transferred to Eppendorf tubes andsonicated for 5 minutes to reduce viscosity. Samples were heated for 10minutes at 70° C. and then loaded onto 4-12% NuPAGE® Bis-Tris gels.BLUelf Pre-stained Protein Ladder (FroggaBio, PM008-0500) was includedas a protein sizing standard. Gels were electrophoresed at 200 Volts for˜1 hour under reducing conditions using 1×MES SDS Running Buffer(Invitrogen, NP0002). Proteins were then transferred to nitrocelluloseusing NuFAGE® Transfer Buffer (Invitrogen, NP0006) plus 20% methanolunder reducing conditions. Blotting was performed for 1 hour at 30Volts. After blotting, membranes were blocked with 5% Blotto (ChemCruz,DC2324) in Tris-Buffered Saline plus Tween (TBST: 10 mM Tris pH 8.0, 150mM NaCl, 0.1% Tween 20) for 1 hour at room temperature with shaking (100rpm). Blots were then probed with primary antibody anti-CT83 rabbitpolyclonal (Sigma, HPA004773) in TBST-5% Blotto at 4 μg/mL overnight at4° C. The next day, blots were washed 5× with TBST and then probed witha 1:5,000 dilution of anti-rabbit IgG HRP conjugated antibody (SouthernBiotech, 4030-05) in TBST-5% Blotto for 1 hour at room temperature withshaking. Blots were washed 5× with TBST and developed by the addition of1-Step Ultra TMB Blotting Solution (Pierce, #37574) (FIG. 47).

Example 24: Expression of Immunostimulatory Factors in A549 and NCI-H460with Reduced Expression of Immunosuppressive Factors

The reduction of immunosuppressive suppressive factors in the VME canenhance cellular immune responses. Expression of immunostimulatoryfactors in the VME, in the context of reduced production ofimmunosuppressive factors, should further enhance the ability of thevaccine to elicit robust immune responses.

In this Example, the A549 and NCI-H460 component vaccine cell lines withreduced expression of three immunosuppressive factors were modified tosecrete GM-CSF, express membrane bound CD40L, and/or secrete thefunctional heterodimeric IL-12 p70 cytokine. The ability for GM-CSF toincrease IFNγ responses in vitro is described in Example 16. In vivoexpression of GM-CSF in the skin enhances DC activation, maturation, andthe ability for DCs to promote a more functional, Th1-biased immuneresponse. The immunostimulatory functions of membrane bound CD40L andIL-12 p70 when expressed alone are described in Example 15 and Example17, respectively. The methods used for shRNA mediated knockdown TGFβ1and TGFβ2 secretion, and to determine resulting secretion levels, aredescribed in Example 5. The methods used for ZFN-mediated knockout ofCD47 and CD276, and to determine resulting cell surface expressionlevels, are described in Example 12 and Example 13, respectively.

In some examples, the component vaccine cell lines with three reducedimmunosuppressive factors were modified to secrete GM-CSF and to expressmembrane bound CD40L. In some examples, the component vaccine cell lineswith three reduced immunosuppressive factors were modified to secreteGM-CSF, express membrane bound CD40L, and to secrete the functionalIL-12 p70 cytokine. Methods used to quantify the expression of membranebound CD40L are described herein.

Secretion of GM-CSF by A549 and NCI-H460

The vaccine component cell lines A549 and NCI-H460 were transduced withlentiviral particles expressing native human GM-CSF. Unmodifiedparental, unmodified cell lines served as controls. After antibioticselection in 100 μg/mL to enrich for cells stable expressing GM-CSF,cells were analyzed for GM-CSF secretion by ELISA. The sequence ofGM-CSF used in this example is shown in SEQ ID NO: 6.

Quantification of Secreted GM-CSF

GM-CSF-transduced and unmodified parental cells were plated at 8.33×10⁴cells/well in a 24-well plated in regular growth medium (RPMI containing10% FBS). Twenty-four hours after plating, adherent cells werethoroughly washed to remove FBS and culture was continued in RPMI+5%CTS. Forty-eight hours after media replacement, the cell culturesupernatant was harvested, and stored at −70° C. until the GM-CSFsecretion assay was completed according to the manufacturersspecifications (human GM-CSF Quantikine ELISA kit #DGM00, R&D Systems).The lower limit of quantitation of human GM-CSF in the ELISA assay isless than 3.0 μg/mL, or 0.018 ng/10⁶ cells/24 hours. GM-CSF secretion bythe unmodified parental cell lines was below the lower limit ofquantitation of the ELISA assay.

Quantification of Secreted IL-12 p70

IL-12-transduced and unmodified parental cells were plated at 8.33×10⁴cells/well in a 24-well plated in regular growth medium (RPMI containing10% FBS). Twenty-four hours after plating, adherent cells werethoroughly washed to remove FBS and culture was continued in RPMI+5%CTS. Forty-eight hours after media replacement, the cell culturesupernatant was harvested, and stored at −70° C. until the IL-12secretion assays for p40 and p70 were completed according to themanufacturers specifications (BioLegend, human IL-12 p40 LEGEND MAXELISA kit #430707 and human IL-12 p70 LEGEND MAX ELISA kit #431707). Thelower limit of quantification of human IL-12 p40 is 9.5 μg/mL, or 0.057ng/10⁶ cells/24 hours. The lower limit of quantification of human IL-12p70 is 1.2 μg/mL, or 0.007 ng/10⁶ cells/24 hours. IL-12 secretion by theunmodified parental cell lines was below the lower limit of quantitationof the ELISA assay.

GM-CSF secretion and membrane bound CD40L expression by TGFβ1 TGFβ2 KDCD47 KO A549 and NCI-H460 cell lines

The A549 cell line was modified to reduce secretion of TGFβ1 86% (n=2)(FIG. 48A) (Table 32), and TGFβ2>89% (n=2) (FIG. 48B) (Table 32), reducethe expression of CD47 99.9% (FIG. 48C) (Table 33), secrete 2,656±69ng/10⁶ cells/24 hours of GM-CSF (FIG. 48D) (Table 34), and express a38-fold increase in membrane bound CD40L (FIG. 48E) (Table 34). TheNCI-H460 cell line was modified to reduce secretion of TGFβ1>95% (n=2)(FIG. 49A) (Table 32), and TGFβ2 93% (n=2) (FIG. 49B) (Table 32), reducethe expression of CD47 99.9% (FIG. 49C) (Table 33), secrete 940±19ng/10⁶ cells/24 hours of GM-CSF (FIG. 49D) (Table 35), and express a5-fold increase in membrane bound CD40L (FIG. 49E) (Table 34).

TABLE 32 TGFβ1 and TGFβ2 secretion in CD47 KO cell lines that secreteGM-CSF and express membrane bound CD40L TGFβ1 (pg/10⁶ cells/24 hours)TGFβ2 (pg/10⁶ cells/24 hours) Cell line Parental TGFβ1 KD % ReductionParental TGFβ2 KD % Reduction A549 4,767 ± 300 679 + 51 86 732 ± 14 <42 >89* NCI-H460 1,850 ± 1  <92 >95* 3,433 ± 271   239 ± 13 93 Parentalindicates the unmodified cell line *Secretion levels are below the lowerlimit of quantification for TGFβ1 (92 pg/10⁶ cells/24 hours) or TGFβ2(42 pg/10⁶ cells/24 hours). Lower limit of quantification used toapproximate % reduction relative to parental. NA: secretion levels arebelow the lower limit of quantification for both the parental and shRNAmodified cell line.

TABLE 33 CD47 KO or CD276 KO in TGFβ1 and TGFβ2 KD cell lines thatsecrete GM-CSF and express membrane bound CD40L Parental Modified Cellline CD47 MFI CD47 MFI % Reduction A549 100,228 74 99.9 NCI-H460 140,99030 99.9 Modified Cell line Parental MFI CD276 MFI % Reduction A54930,636 1,983 93.5 NCI-H460 82,858 712 99.1 MFI reported with unstainedcontrols subtracted. Parental indicates the unmodified cell line.

TABLE 34 GM-CSF secretion and membrane bound CD40L expression by TGFβ1TGFβ2 KD CD47 KO and TGFβ1 TGFβ2 KD CD276 KO cell lines GMCSF ParentalModified CD40L (ng/10⁶ cells/ CD40L CD40L Fold Cell line 24 hours) MFIMFI Increase A549 TGFβ1 2,656 ± 69    9,537   360,236 38 and TGFβ2 KD,CD47 KO NCI-H460 TGFβ1 940 ± 19  16,992   84,924  5 and TGFβ2 KD, CD47KO A549 TGFβ1 1,704 ± 60   41,076 1,660,242 40 and TGFβ2 KD, CD276 KONCI-H460 TGFβ1 943 ± 13  16,992   121,555  7 and TGFβ2 KD, CD276 KO

GM-CSF Secretion and Membrane Bound CD40L Expression by TGFβ1 TGFβ2 KDCD276 KO A549 and NCI-H460 Cell Lines

The A549 cell line was modified to reduce secretion of TGFβ1>98% (n=2)(FIG. 50A) (Table 35), and TGFβ2>89% (n=2) (FIG. 50B) (Table 35), reducethe expression of CD276 93.5% (FIG. 50C) (Table 33), secrete 1,704±60ng/10⁶ cells/24 hours of GM-CSF (FIG. 50D) (Table 34), and express a40-fold increase in membrane bound CD40L (FIG. 50E) (Table 34). TheNCI-H460 cell line was modified to reduce secretion of TGFβ1 93% (n=2)(FIG. 51A) (Table 32), and TGFβ2 89% (n=2) (FIG. 51B) (Table 32), reducethe expression of CD276 99.1% (FIG. 51C) (Table 33), secrete 943±13ng/10⁶ cells/24 hours of GM-CSF (FIG. 51D) (Table 34), and express a7-fold increase in membrane bound CD40L (FIG. 51D) (Table 34).

TABLE 35 TGFβ1 and TGFβ2 secretion in CD276 KO cell lines that secreteGM-CSF and express membrane bound CD40L TGFβ1 (pg/10⁶ cells/24 hours)TGFβ2 (pg/10⁶ cells/24 hours) Cell line Parental TGFβ1 KD % ReductionParental TGFβ2 KD % Reduction A549 4,967 ± 399 <92 >98*  807 ± 8 <42 >89* NCI-H460 1,850 ± 1  126 ± 5 93 3,433 ± 271 366 ± 5 89 Parentalindicates the unmodified cell line. *Secretion levels are below thelower limit of quantification for TGFβ1 (92 pg/10⁶ cells/24 hours) orTGFβ2 (42 pg/10⁶ cells/24 hours). Lower limit of quantification used toapproximate % reduction relative to parental.

GM-CSF Secretion and Membrane Bound CD40L Expression by TGFβ1 TGFβ2 KDCD47 KO and TGFβ1 TGFβ2 KD CD276 KO A549 Cell Line Increases CellularImmune Responses

IFNγ ELISpot was used to evaluate the effect GM-CSF secretion andmembrane bound CD40L expression by TGFβ1 TGFβ2 KD CD47 KO and GM-CSFsecretion and membrane bound CD40L expression by TGFβ1 TGFβ2 KD CD276 KOon cellular immune responses in the A549 cell line. IFNγ ELISpot wascompleted as described in Example 9 using cells derived from two HLA-A02healthy donors (n=3/donor). GM-CSF secretion and membrane bound CD40Lexpression by TGFβ1 TGFβ2 KD CD47 KO (3,213±287) (n=6) (p=0.0357) andTGFβ1 TGFβ2 KD CD276 KO (3,207±663) (n=6) (p=0.0143) significantlyincrease IFNγ responses compared to the unmodified parental A549 cellline (1,793±215 SFU) (n=6) (FIG. 52A). Statistical significance wasdetermined using One-Way ANOVA and Holm-Sidak's multiple comparisonstest.

GM-CSF Secretion and Membrane Bound CD40L Expression by TGFβ1 TGFβ2 KDCD276 KO A549 and NCI-H460 Cell Lines Increase DC Maturation

The maturation of iDCs was determined by flow cytometry as described inExample 15. In this Example, iDCs derived from three HLA-A02 donors wereco-cultured with the unmodified parental A549 or unmodified parentalNCI-H460 cell lines, or the modified A549 or NCI-H460 TGFβ1 and TGFβ2 KDCD276 KO, that secrete GM-CSF and express membrane bound CD40L.Expression of the DC maturation marker CD83 was significantly increasedon DCs co-cultured with the modified A549 (71±2%) compared to DCsco-cultured with the unmodified parental A549 cell line (53±3%)(p=0.0015) (FIG. 52B). Similarly, CD83 was significantly increased onDCs co-cultured with the modified NCI-H460 (71±5%) compared to DCsco-cultured with the unmodified parental H460 (ATCC HTB-177) cell line(52±3%) (p=0.0126) (FIG. 52C). Statistical significance was determinedusing One-Way ANOVA and Holm-Sidak's multiple comparisons test.

GM-CSF Secretion, Membrane Bound CD40L Expression, and IL-12 Secretionby TGFβ1 TGFβ2 KD CD47 KO A549 and NCI-H460 Vaccine Component Cell Lines

The A549 cell line was modified to reduce secretion of TGFβ1 84% (n=2)(FIG. 53A) (Table 36), and TGFβ2>89% (n=2) (FIG. 53B) (Table 36), reducethe expression of CD47 99.9% (FIG. 53C) (Table 33), secrete 2,295±60ng/10⁶ cells/24 hours of GM-CSF (FIG. 53D) (Table 37), express a 56-foldincrease in membrane bound CD40L (FIG. 53E) (Table 37), and secrete300±24 ng/10⁶ cells/24 hours of IL-12 p70 (FIG. 53F) (Table 37).

TABLE 36 TGFβ1 and TGFβ2 secretion in TGFβ1 and TGFβ KD, CD47 KO celllines that secrete GM-CSF, express membrane bound CD40L, and secreteIL-12 TGFβ1 (pg/10⁶ cells/24 hours) TGFβ2 (pg/10⁶ cells/24 hours) Cellline Parental TGFβ1 KD % Reduction Parental TGFβ2 KD % Reduction A5494,767 ± 300 760 ± 55 84  732 ± 14 <42 >89* NCI-H460 1,850 ± 1  <92 >95*3,433 ± 271 492 ± 10 86 Parental refers to the unmodified cell line.*Secretion levels are below the lower limit of quantification for TGFβ1(92 pg/10⁶ cells/24 hours) or TGFβ2 (42 pg/10⁶ cells/24 hours). Lowerlimit of quantification used to approximate % reduction relative toparental.

TABLE 37 GM-CSF secretion, membrane bound CD40L expression, and IL-12secretion by TGFβ1 TGFβ2 KD CD47 KO and TGFβ1 TGFβ2 KD CD276 KO celllines GMCSF IL-12 (ng/ Parental Modified CD40L p70 (ng/ 10⁶ cells/ CD40LCD40L Fold 10⁶ cells/ Cell line 24 hours) MFI MFI Increase 24 hours)A549 TGFβ1 2,295 ± 60  9,537   536,953 56 300 ± 24 and TGFβ2 KD, CD47 KONCI-H460 TGFβ1 1,586 ± 24 16,992   154,964  9 434 ± 15 and TGFβ2 KD,CD47 KO A549 TGFβ1 1,113 ± 51 41,076 1,476,699 36 263 ± 24 and TGFβ2 KD,CD276 KO NCI-H460 TGFβ1 1,234 ± 24 16,992   267,023 16 312 ± 50 andTGFβ2 KD, CD276 KO

The NCI-H460 cell line was modified to reduce secretion of TGFβ1>95%(n=2) (FIG. 54A) (Table 36), and TGFβ2 86% (n=2) (FIG. 54B) (Table 36),reduce the expression of CD47>99.9% (FIG. 54C) (Table 33), secrete1,586±24 ng/10⁶ cells/24 hours of GM-CSF (FIG. 54C) (Table 37), expressa 9-fold increase in membrane bound CD40L (FIG. 54E) (Table 36), addsecrete 434±15 ng/10⁶ cells/24 hours of IL-12 p70 (FIG. 54F) (Table 36).

GM-CSF Secretion, Membrane Bound CD40L Expression, and IL-12 Secretionby TGFβ1 TGFβ2 KD CD47 KO A549 (ATCC CCL-185) and NCI-H460 (ATCCHTB-177) Cell Lines Increases TAA-Specific IFNγ Responses

IFNγ ELISpot was used to evaluate the effect GM-CSF secretion,expression of membrane bound CD40L, and secretion of IL-12 by the TGFβ1TGFβ2 KD CD47 KO A549 and by the TGFβ1 TGFβ2 KD CD47 KO NCI-H460 celllines on IFNγ responses to antigens. IFNγ ELISpot was completed asdescribed in Example 9 using cells derived from two HLA-A02 healthydonors (n=3/donor). The total IFNγ response to the TAAs MAGE A3,Survivin, PRAME, Muc1, STEAP1, Her2, and TERT was increased by the A549TGFβ1 TGFβ2 KD CD47 KO cells (1,586±887 SFU) (n=6) compared to theunmodified parental cell line (382±96 SFU) (n=6) (p=0.5887) (FIG. 55A).Similarly, the total antigen specific IFNγ response elicited by theNCI-H460 TGFβ1 TGFβ2 KD CD47 KO cell line (1702±682 SFU) (n=6) wasincreased relative to the unmodified parental cell line (262±105 SFU)(n=6) (p=0.1385) (FIG. 55B). Responses to specific antigens are in theorder indicated in the figure legends.

GM-CSF Secretion, Membrane Bound CD40L Expression, and IL-12 Secretionby TGFβ1 TGFβ2 KD CD276 KO A549 and NCI-H460 Vaccine Component CellLines

The A549 cell line was modified to reduce the secretion of TGFβ1 96%(n=2) (FIG. 56A) (Table 38), and TGFβ2>89% (n=2) (FIG. 56B) (Table 38),reduce the expression of CD276 98.9% (FIG. 56C) (Table 33), secrete1,113±51 ng/10⁶ cells/24 hours of GM-CSF (FIG. 56D) (Table 37), expressa 36-fold increase in membrane bound CD40L (FIG. 56E) (Table 37), addsecrete 263±24 ng/10⁶ cells/24 hours of IL-12 p70 (FIG. 56F) (Table 37).

NCI-H460 cell line was modified to reduce secretion of TGFβ1>95% (n=2)(FIG. 57A) (Table 38), and TGFβ2 78% (n=2) (FIG. 57B) (Table 38), reducethe expression of CD276 98.2% (FIG. 57C) (Table 33), secrete 1,234±24ng/10⁶ cells/24 hours of GM-CSF (FIG. 57D) (Table 37), express a 16-foldincrease in membrane bound CD40L (FIG. 57E) (Table 37), add secrete312±50 ng/10⁶ cells/24 hours of IL-12 p70 (FIG. 57F) (Table 37).

TABLE 38 TGFβ1 and TGFβ2 secretion in cell lines with reduced CD276expression modified to express CD40L, GM-CSF, and IL-12 p70 TGFβ1(pg/10⁶ cells/24 hours) TGFβ2 (pg/10⁶ cells/24 hours) Cell line ParentalTGFβ1 % Reduction Parental TGFβ2 % Reduction A549 4,967 ± 399 179 ± 6 96 807 ± 8  <42 >89* NCI-H460 1,850 ± 1  <92 >95* 3,433 ± 271 738 ± 34 78Parental indicates the unmodified cell line. *Secretion levels are belowthe lower limit of quantification for TGFβ1 (92 pg/10⁶ cells/24 hours)or TGFβ2 (42 pg/10⁶ cells/24 hours). Lower limit of quantification usedto approximate % reduction relative to parental. NA: secretion levelsare below the lower limit of quantification for both the parental andshRNA modified cell line.

GM-CSF Secretion, Membrane Bound CD40L Expression, and IL-12 Secretionby TGFβ1 TGFβ2 KD CD276 KO A549 and NCI-H460 Cell Lines Increases DCMaturation

The effect of GM-CSF secretion, expression of membrane bound CD40L, andsecretion of IL-12 by the component vaccine cell lines on the maturationof DCs was determined by flow cytometry as described in Example 15.Specifically, iDCs derived from three HLA-A02 donors were co-culturedwith the unmodified parental A549 (ATCC CCL-185) or NCI-H460 (ATCCHTB-177) cell lines, or the modified TGFβ1 and TGFβ2 KD CD276 KO A549(ATCC CCL-185) or NCI-H460 (ATCC HTB-177) that secrete GM-CSF, expressmembrane bound CD40L, and secrete IL-12. Expression of the DC maturationmarker CD83 was significantly increased on DCs co-cultured with themodified A549 (ATCC CCL-185) (71±3%) cell line compared to DCsco-cultured with the unmodified parental A549 (ATCC CCL-185) cell line(53±3%) (p=0.0014) (FIG. 58A). Similarly, CD83 was significantlyincreased on DCs co-cultured with the modified NCI-H460 (69±4%) cellline compared to DCs co-cultured with the unmodified parental H460 (ATCCHTB-177) cell line (52±3%) (p=0.0077) (FIG. 58B). Statisticalsignificance was determined using One-Way ANOVA and Holm-Sidak'smultiple comparisons test.

GM-CSF Secretion, Membrane Bound CD40L Expression, and IL-12 Secretionby TGFβ1 TGFβ2 KD CD276 KO A549 (ATCC CCL-185) and NCI-H460 (ATCCHTB-177) Cell Lines Increases TAA-Specific IFNγ Responses

IFNγ ELISpot was used to evaluate the effect GM-CSF secretion,expression of membrane bound CD40L, and secretion of IL-12 by the TGFβ1TGFβ2 KD CD276 KO A549 and by the TGFβ1 TGFβ2 KD CD276 KO NCI-H460 celllines on IFNγ responses to antigens. IFNγ ELISpot was completed asdescribed in Example 9 using cells derived from two HLA-A02 healthydonors (n=3/donor). The total IFNγ response to the antigens MAGE A3,Survivin, PRAME, Muc1, STEAP1, Her2, and TERT was markedly increased bythe A549 TGFβ1 TGFβ2 KD CD47 KO cells (1,408±738 SFU) (n=6) compared tothe unmodified parental cell line (421±149 SFU) (n=6) (p=0.1385) (FIG.58C). Similarly, the total antigen specific IFNγ response elicited bythe NCI-H460 TGFβ1 TGFβ2 KD CD276 KO cell line (1725±735 SFU) (n=6) wasincreased relative to the unmodified parental cell line (262±105 SFU)(n=6) (p=0.1385) (FIG. 58D). Responses to specific antigen are in theorder indicated in the figure legends.

Example 25: HLA Mismatch Results in Increased Immunogenicity

Immune cells respond to “non-self”-proteins by generating an immuneresponse. In the case of HLA mismatch, the immune response is againstHLA proteins that are not expressed on the individual's cells and thisresponse can be measured by the production of interferon gamma.Interferon gamma is a key cytokine involved in the generation of a Th₁ Tcell response and Th₁ T cells are the essential mediators of ananti-cancer response. Unlike in stem cell or organ transplants, the HLAmismatch immune response plays a highly beneficial role in increasingthe immunogenicity of a whole cell tumor vaccine by acting as anadjuvant that boosts the priming of T cells to TAAs expressed within thetumor vaccine.

According to various embodiments of the present disclosure, the designof a cocktail of cell lines comprising the final vaccine product toinclude HLA mismatches at the two most immunogenic HLA loci-HLA-A andHLA-B, between the vaccine and the patient results in beneficialinflammatory responses at the vaccine site that results in increasedvaccine uptake and presentation by DCs and the activation of a largernumber of T cells, thus ultimately increasing the breadth, magnitude andimmunogenicity of tumor reactive T cells primed by the cancer vaccinecocktail. By including multiple cell lines chosen to have mismatches inHLA types, and chosen for expression of key TAAs, the vaccine enableseffective priming of a broad and effective anti-cancer response with theadditional adjuvant effect generated by the HLA mismatch.

In one example, a vaccine composition according to the presentdisclosure includes multiple cell lines chosen to ensure a breadth ofTAAs as well as a diversity in the most immunogenic HLA proteins (HLA-Aand HLA-B) in order to stimulate a maximal, effective immune responseagainst the tumor. Inclusion of HLA mismatch augments the immuneresponse, acting as an adjuvant to result in increased total anti-TAAinterferon gamma production measurable by ELISpot and flow cytometry.The following features and selection criteria can be followed accordingto various embodiments:

Since HLA genes are inherited, the degree of HLA mismatch increasesamongst individuals from different ethnicities. The cell line selectionprocess may thus include, in some embodiments, obtaining cells frombanks around the world in order to design a cocktail to includediversity in HLA alleles.

Disparities in HLA-C, -DRB1 and -DPB1 have been identified to bepotentially less immunogenic, therefore in some embodiments the celllines of a vaccine composition may be selected to ensure a mismatch ofat least 2 of the highly immunogenic HLA-A and HLA-B alleles.

Increasing the number of mismatched HLA-A and HLA B loci between thecell lines selected may result, according to some embodiments, in agreater degree of mismatch across all patients receiving the vaccine toensure the adjuvant effect measurable by interferon gamma ELISpot.

Dendritic cells were incubated with cancer cell line to allow forantigen uptake and DC maturation. The DCs were then co-cultured withPBMCs from donors, re-stimulated with the same cell line or a cocktailof cell lines chosen to have heterogeneity in their HLA subtypes and inorder to create a mismatch with the donor PBMC HLA type. The cells wereplated on an ELISpot plate and activated. Tumor specific T cells weremeasured by counting interferon γ spots/well as described in Example 6.

As shown in FIG. 59, inclusion of a combination of lung cancer celllines with a greater degree of HLA mismatch to the donor across multipleHLA molecules results in increased anti-tumor T cell responses. Theimmune response due to HLA mismatch acts as an adjuvant to boost overallresponses. These data indicated that inclusion of multiple cell lines toensure a broad degree of HLA mismatch on multiple class I and class IIHLA molecules between whole tumor cancer vaccine cocktail and recipientcan generate an increased allogeneic response.

Example 26: Preparation of Non-Small Cell Lung Cancer (NSCLC) Vaccines

Tumors and tumor cell lines are highly heterogeneous. The subpopulationswithin the tumor express different phenotypes with different biologicalpotential and different antigenic profiles. One of the driving purposesbehind a whole tumor cell vaccine is to present a wide array of tumorcells to the immune system. By doing this, the immune response isgenerated against multiple TAAs, bypassing issues related to antigenloss, which can lead to antigen escape (or immune relapse) and patientrelapse (Keenan B P, et al., Semin Oncol. 2012; 39: 276-86). Antigenescape was first observed in the treatment of B-cell lymphoma withanti-idiotype monoclonal antibodies (Meeker T, et al., N Engl J Med.1985; 312: 1658-65) and has since been observed in other immunotherapytreatments such as CAR-T therapy (Majzner R G, et al., Cancer Discov.2018; 8: 1219-26).

Expression of NSCLC TAAs

Expression of twenty-four TAAs by candidate component cell lines wasdetermined by RNA expression data sourced from Broad Institute CancerCell Line Encyclopedia (CCLE). The HGNC gene symbol was included in theCCLE search and mRNA expression was downloaded for each TAA. Expressionof a TAA by a cell line was considered positive if the RNA-seq value(FPKM) was greater than 0.5. Collectively, the six component cell linesexpressed twenty-three of the twenty-four identified TAAs at a mRNAlevel>0.5 FPKM (FIG. 60). Specifically, five TAAs were expressed by onecell line, four TAAs were expressed by two cell lines, four TAAs wereexpressed by three cell lines, five TAAs were expressed by three celllines, and six TAAs were expressed by eight cell lines. The minimumnumber of TAAs expressed by a single cell line was twelve (NCI-H520) andthe maximum number of TAAs expressed by a single cell was eighteen (DMS53). The number of antigens that can be targeted by the exemplary 6-cellline unit dose comprised of A549, NCI-H520, NCI-H460, DMS 53, LK-2,NCI-H23 is higher than the individual cell lines.

The cells in the vaccine described herein were selected to express awide array of TAAs, including those known to be important to antitumorimmunity. To further enhance the array of TAAs, one cell line (LK-2) wasalso transduced with the genes for CT83 and mesothelin, as describedherein (FIG. 65). CT83 mRNA was endogenously expressed at a low level intwo of the six cell lines and mesothelin was endogenously expressed byone of the six component cell lines.

Because of the need to maintain maximal heterogeneity of TAAs, the genemodified cell lines utilized in the present vaccine have beenestablished using antibiotic selection and flow cytometry and notthrough limiting dilution subcloning.

Cumulatively, the cells in the present vaccine express more of the TAAsthat have been demonstrated to be important in antitumor immunity. Thecell lines in Table 39 are used in the present NSCLC vaccine.

TABLE 39 NSCLC vaccine cell lines and histology Cocktail Cell Line NameHistology A NCI-H520 Squamous A A549 Adenocarcinoma A NCI-H460 Largecell B LK-2 Squamous B NCI-H23 Adenocarcinoma B DMS 53 SCLC

shRNA Downregulates TGF-β Secretion

TGFβ1 and TGFβ2 was knocked down and resulting secretion levelsdetermined as described in Example 5. Of the parental cell lines inCocktail A, NCI-H460 and A549 secrete measurable levels of TGFβ1 andTGFβ2 while LK-2 secretes TGFβ2 but not TGFβ1. Of the parental celllines in Cocktail B, NCI-H23 secretes measurable levels of TGFβ1 andTGFβ2 and LK-2 secretes TGFβ2 but not TGFβ1. DMS 53 secretes measurablelevels of TGFβ1 and TGFβ2, but TGFβ1 secretion is low.

With the exception of DMS 53, the component cell lines were alltransduced with TGFβ1 shRNA and TGFβ2 shRNA to knockdown secretion ofthe two molecules. DMS 53 was gene modified with TGFβ2 shRNA onlybecause multiple attempts to modify with both TGFβ1 and TGFβ2 shRNA werenot successful. TGFβ1 knockdown was chosen to move forward because thesecretion levels of TGFβ2 were already low in this cell line. Thesecells are described by the clonal designation DK4. The remaining celllines were double modified with TGFβ1 and TGFβ2 shRNA. These cells aredescribed by the clonal designation D K6.

Table 40 shows the TGF-β secretion in gene modified component cell linescompared to wild type cell lines. Reduction of TGFβ1 ranged from 59% to90%. Reduction of TGFβ2 ranged from 42% to 97%.

TABLE 40 TGF-β Secretion (pg/10⁶ cells/24 hr) in Component Cell LinesCell Line Cocktail Clone TGFβ1 TGFβ2 NCI-H520 A Wild type ND 3872NCI-H520 A DK6 ND  124 NCI-H520 A Percent reduction NA 97% A549 A Wildtype 5727  775 A549 A DK6  577   42 A549 A Percent reduction 90% 95%NCI-H460 A Wild type 1573 2307 NCI-H460 A DK6  287  533 NCI-H460 APercent reduction 82% 77% LK-2 B Wild type ND  161 LK-2 B DK6 ND   69LK-2 B Percent reduction NA 88% NCI-H23 B Wild type 1761  588 NCI-H23 BDK6  719   61 NCI-H23 B Percent reduction 59% 90% DMS 53 B Wild type 261 2833 DMS 53 B DK4  286 1640 DMS 53 B Percent reduction  0% 42% DK6:TGFβ1/TGFβ2 double knockdown; DK4: TGFβ2 single knockdown; ND = notdetectable; NA = not applicable

Based on an injected dose of 8×10⁶ of each component cell line, thetotal TGF-β secretion in Cocktails A and B is shown in Table 41.Secretion in the wild type cells in the cocktail is also shown. CocktailA shows a total secretion of 9679 μg per injected dose per 24 hours forTGFβ1 and 5600 μg per injected dose per 24 hours for TGFβ2. Cocktail Bshows a total secretion of 8220 μg per injected dose per 24 hours forTGFβ1 and 14163 μg per injected dose per 24 hours for TGFβ2.

Belagenpumatucel-L had a total TGFβ2 secretion of 18,813 μg per injecteddose per 24 hours (Nemunaitis, J. et al. JCO. (2006) 24:29, 4721-4730)(Fakhrai, H 2010). The total TGFβ2 secretion in the NSCLC vaccine(19,763 μg per injected dose per 24 hours) is roughly equivalent to theTGFβ2 secretion in belagenpumatucel-L despite the higher injected cellnumber of 4.8×10⁷ cells in the NSCLC vaccine compared to 2.5×10⁷ cellsin belagenpumatucel-L.

TABLE 41 Total TGF-β Secretion (pg/dose/24 hr) in NSCLC vaccineCocktails Cocktail Clones TGFβ1 TGFβ2 A Wild type 58592 55638 K6  9679 5600 Percent reduction 83% 90% B Wild type 16735 28654 DK6/4  822014163 Percent reduction 51% 51%

The total TGFβ1 secretion in the NSCLC vaccine (17,899 μg per injecteddose per 24 hours) is 31% of the estimated TGFβ1 secretion inbelagenpumatucel-L.

CD276 Expression

All component cell lines expressed CD276 and CD276 expression wasknocked out by electroporation with ZFN as described in Example 13 andherein. The component cell lines had previously been gene modified withshRNA to knockdown expression of TGFβ1 and TGFβ2 (termed DK6), apartfrom DMS 53, where only TGFβ2 was knocked down (termed DK4). Because itwas desirable to maintain as much tumor heterogeneity as possible, theelectroporated cells were not cloned by limiting dilution. Instead, thecells were subjected to multiple rounds of cell sorting by FACS.Reduction of CD276 expression is described in Table 42. The absence ofprotein expression in the knockout cells was also confirmed by westernblot analysis using (data not shown). These data show that gene editingof CD276 resulted in greater than 99% CD276-negative cells in all sixcomponent cell lines.

TABLE 42 Reduction of CD276 expression Parental Cell TGβ1/B2 KD %Reduction Cell line Line MFI CD276 KO MFI CD276 NCI-H460 366,565 83899.8 NCI-H520 341,202 212 99.9 A549 141,009 688 99.5 DMS 53 304,637 97299.7 LK-2 385,535 867 99.8 NCI-H23  74,176 648 99.1 MFI reported withunstained controls subtracted

GM-CSF Secretion

Component cell lines were transduced with the GM-CSF as described hereinand Example 24. The results are shown in Table 43.

TABLE 43 GM-CSF Secretion in Component Cell Lines GM-CSF GM-CSF CellLine (ng/10⁶ cells/24 hr) (ng/dose/24 hr) NCI-H520 10 80 A549 288023,040 NCI-H460 1330 10,640 Cocktail A Total 4220 33,760 LK-2 2 16NCI-H23 2310 18,480 DMS 53 170 1,360 Cocktail B Total 2482 19,856

Based on an injected dose of 8×10⁶ of each component cell line, thetotal GM-CSF secretion for Cocktail A is 33,760 ng per injected dose per24 hours. The total GM-CSF secretion for Cocktail B is 19,856 ng perinjected dose per 24 hours. The total secretion per injection istherefore 43,616 ng per 24 hours.

CD40L Expression

The component cell lines were transduced with a CD40L vector asdescribed herein and by the methods described in Example 15. CD40Lexpression was evaluated by flow cytometry with an anti-CD40L monoclonalantibody as described in Example 15. The results, shown in FIG. 74,demonstrated significant CD40L membrane expression in all six celllines.

IL-12 Expression

The component cell lines were transduced with the IL-12 vector andresulting IL-12 p70 expression determined as described in Example 24 andherein the results are shown in Table 44.

TABLE 44 IL-12 secretion in component cell lines IL-12 IL-12 Cell Line(ng/10⁶ cells/24 hr) (ng/dose/24 hr) NCI-H520 NA NA A549 440 3520NCI-H460 420 3360 Cocktail A Total 860 6880 LK-2 NA NA NCI-H23 580 4640DMS 53 140 1120 Cocktail B Total 720 5760

Based on an injected dose of 8×10⁶ of each component cell line, thetotal IL-12 secretion for Cocktail A is 6880 ng per injected dose per 24hours. The total IL-12 secretion for Cocktail B is 5760 ng per injecteddose per 24 hours. The total IL-12 secretion per injection is therefore12,640 ng per 24 hours.

Stable Expression of Mesothelin and CT83 by the LK-2 Cell Line

As described above, the cells in the vaccine described herein wereselected to express a wide array of TAAs, including those known to beimportant to antitumor immunity. To further enhance the array ofantigens, the LK-2 cell line that was modified to reduce the secretionof TGFβ2, reduced the expression of CD276, and to express GM-CSF andmembrane bound CD40L was also transduced with lentiviral particlesexpressing the CT83 and Mesothelin antigens. The CT83 and mesothelinantigens are linked by a P2A cleavage site (SEQ ID NO: 21).

The expression of membrane bound Mesothelin and CT83 was characterizedby flow cytometry. Unmodified parental and modified cells were stainedextracellular with anti-mesothelin-PE (R&D Systems FAB32652P) accordingto the manufacturers instructions. Unmodified parental and modifiedcells were stained intracellular with anti-CT83 (Abcam, ab121219)followed by goat anti-rabbit Alex488 (Invitrogen, A-11034). The MFI ofthe unstained unmodified parental cells was subtracted from the MFI ofthe stained unmodified cells for both CT83 and mesothelin. The MFI ofthe modified parental cells was subtracted from the MFI of the modifiedcells for both CT83 and mesothelin. Percent increase in expression iscalculated as: (1−(background subtracted modified MFI/backgroundsubtracted unmodified MFI))×100). Expression of CT83 increased in themodified cell line (934,985 MFI) 3-fold over that of the parental cellline (323,878 MFI). Expression of mesothelin by the modified cell line(123,128 MFI) increased 85-fold over the that of the parental cell line(1443 MFI) (FIG. 65A).

IFNγ responses to the CT83 and mesothelin antigens were determined byautologous DC and CD14-PBMC co-culture followed by ELISpot as describedin Example 8. IFNγ responses to the CT83 and mesothelin antigensexpressed by the modified LK-2 cell line were evaluated in the contextof the NSCLC-vaccine B. Specifically, 5×10⁵ of the modified DMS 53,NCI-H23, and LK-2 cells, 1.5×10⁶ total modified cells, were co-culturedwith 1.5×10⁶ iDCs from 3 HLA diverse donors (n=3/donor). CD14-PBMCsisolated from co-culture with mDCs on day 6 were stimulated with theCT83 and mesothelin peptide pools, 15-mers overlapping by 11 amino acidsspanning the native protein sequences, in the IFNγ ELISpot assay for 24hours prior to detection of IFNγ SFU. IFNγ production was detected toboth CT83 (205±158 SFU) (n=9) and mesothelin (3449±889 SFU) (n=9) (FIG.65B).

Vaccine Cocktails Elicited Stronger and Broader Cellular ImmuneResponses Compared to Individual Component Cell Lines

The ability of the individual NSCLC vaccine component cell lines toinduce IFNγ responses against themselves compared to the ability of theNSCLC vaccine cocktails to induce IFNγ responses against the individualcell lines was measured by IFNγ ELISpot as described in Examples 8 and9. The data in FIG. 62 demonstrate that the cocktails (NSCLC-A andNSCLC-B) elicited stronger immune responses than the individualcomponent cell lines for 4 of the 6 cell lines.

The immune response induced by the vaccine cocktails against relevantTAAs was then measured. Normal donor PBMCs were co-cultured withindividual component cell lines or with the NSCLC-A or NSCLC-B cocktailsfor 6 days prior to stimulation with autologous DCs loaded withTAA-specific specific peptide pools containing known MHC-I restrictedepitopes. Cells were then assayed for IFNγ secretion in the IFNγ ELISpotassay. The data shown in FIG. 63 demonstrate that each of the NSCLCvaccine component cell lines is capable of inducing TAA-specific IFNγresponses. More importantly, the two NSCLC vaccine cocktails inducedstronger IFNγ responses against more TAAs compared to the individualcomponent cell lines, indicating that the vaccine cocktails were capableof inducing broader immune responses.

Example 27: Non-Small Cell Lung Cancer (NSLC) Vaccines

Based on the disclosure and data provided herein, the following Exampleprovides a whole cell vaccine for NSCLC comprised of the six lung cancercell lines shown below in Table 45. The cell lines represent twoadenocarcinomas (A549 and NCI-H23), two squamous cell carcinomas(NCI-H520 and LK-2), one large cell carcinoma (NCI-H460), and one smallcell lung cancer (SCLC) (DMS 53). The cell lines have been divided intotwo groupings: vaccine cocktail A and vaccine cocktail B (i.e., NSCLC-Aand NSCLC-B). Cocktail A is designed to be administered intradermally inthe upper arm and Cocktail B is designed to be administeredintradermally in the thigh. Cocktail A and B together comprise a unitdose of cancer vaccine.

TABLE 45 Cell line nomenclature and modifications TGFβ1 TGFβ2 CD276Cocktail Cell Line KD KD KO GM-CSF CD40L IL-12 MSLN CT83 A NCI-H520 X XX X X ND ND ND A A549 X X X X X X ND ND A NCI-H460 X X X X X X ND ND BLK-2 X X X X X ND X X B NCI-H23 X X X X X X ND ND B DMS 53 ND X X X X XND ND ND = Not done

Where indicated in the above table, the genes for the immunosuppressivefactors transforming growth factor-beta 1 (TGFβ1) and transforminggrowth factor-beta 2 (TGFβ2) have been knocked down using shRNAtransduction with a lentiviral vector. The gene for CD276 has beenknocked out by electroporation using zinc-finger nuclease (ZFN). Thegenes for granulocyte macrophage—colony stimulating factor (GM-CSF),IL-12, CD40L, mesothelin, and CT83 have been added by lentiviral vectortransduction.

Five of the six established lung cancer cell lines were obtained fromthe American Type Culture Collection (ATCC, Manassas, Va.) and one wasobtained from the Japanese Collection of Research Bioresources cell bank(JCRB, Kansas City, Mo.).

Example 28: Comparison of Belagenpumatucel-L and NSCLC Vaccine

The results of the clinical studies of belagenpumatucel-L were publishedin peer-reviewed journals and included two Phase II trials (NemunaitisJ, et al., J Clin Oncol. 2006; 24: 4721-30; Nemunaitis J, et al., CancerGene Ther. 2009; 16: 620-4) and a Phase III trial (Giaccone G, et al.,Eur J Cancer. 2015; 51: 2321-9) in NSCLC.

Belagenpumatucel-L was a vaccine in which TGFβ2 secretion in fourallogeneic NSCLC tumor cell lines was down-regulated using a TGFβ2antisense plasmid. However, Belagenpumatucel-L did not address the issueof TGFβ1 secretion. Recent studies have shown that TGFβ1 is thepredominant isoform expressed in the immune system. TGFβ1 binds to theTGFβRII receptor at high affinity, whereas TGFβ2 only binds with highaffinity in the presence of the TGFβRIII co-receptor (also calledbetaglycan). Betaglycan is downregulated in NSCLC, which makes TGFβ1 thepredominant TGFβ isoform.

The NSCLC vaccine described in Example 27 introduces great improvementover belagenpumatucel-L relative to secretion of TGFβ1 and TGFβ2, amongother modifications and improvements. The lower level of TGFβ2 secretionin the NSCLC vaccine is important, but even more significant is thedecreased level of TGFβ1. The present NSCLC vaccine also introduces thefollowing improvements: use of lentiviral transduction of shRNA is beingused to knockdown the expression of TGFβ2 and TGFβ1 providing a majorimprovement over antisense for both expression and stability; use ofzinc-finger nuclease electroporation to knockout the expression ofCD276; use of lentiviral transduction to induce expression of theimmunostimulatory molecules GM-CSF, IL-12, and CD40L; use of a SCLC cellline noting recent observations that NSCLC tumors contain a significantSCLC component and that component is responsible for drug resistance,metastasis, and relapse; and use of a serum-free media formulation.

As described above, twenty-four TAAs that could potentially generate arelevant antitumor immune response in NSCLC patients were identified.mRNA expression of these twenty-four antigens in the NSCLC vaccine andbelagenpumatucel-L is shown in FIG. 61A. The data in FIG. 61 isillustrated as the sum of Log₁₀ FPKM+14 mRNA expression of each antigenin the respective belagenpumatucel-L and NSCLC vaccine cell linecomponents. The FPKM mRNA value was adjusted by 14.0 to account for thenegative base value (−13.00 FPKM) to allow for addition of mRNA levelswith positive values. Expression of the twenty-three prioritized NSCLCTAAs expressed by the NSCLC vaccine cell components was determined in573 NSCLC patient samples. The NSCLC patient data was downloaded fromthe publicly available database, cBioPortal (cbioportal.org) (Cerami, E.et al. Cancer Discovery. 2012; Gao, J. et al. Sci Signal. 2013) betweenFeb. 23, 2020 through Jul. 2, 2020 (FIG. 78C). The HUGO GeneNomenclature Committee (HGNC) gene symbol was included in the search andmRNA expression was downloaded for each TAA.

The NSCLC vaccine potentially targets a median of 21 TAAs (FIG. 61B) andbelagenpumatucel-L targets a median of 17 TAAs (FIG. 61C) expressed bythe 573 patient tumor samples. The NSCLC vaccine and belagenpumatucel-Lboth have the potential to induce an antitumor response to at least fiveantigens in all 573 patients. The NSCLC vaccine has the potential toinduce an antitumor response to at least 17 antigens in 572 patients(99.8%), at least 18 antigens in 565 patients (98.6%), at least 19antigens in 538 patients (93.9%), at least 20 antigens in 438 patients(76.4%), at least 21 antigens in 290 patients (50.6%), at least 22antigens in 183 patients (31.9%) and at least 23 antigens in 73 patients(12.7%). In comparison, belagenpumatucel-L could only induce anantitumor response to at least 14 antigens in 572 patients (99.8%), atleast 15 antigens in 558 patients (97.4%), at least 16 antigens in 525patients (91.6%), at least 17 antigens in 351 patients (61.3%), at least18 antigens in 233 patients (40.7%) and at least 19 antigens in 126patients (22.0%). The above analysis includes antigens prioritized toinduce and antitumor response in NSCLC patients and does not account forthe additional, and potentially clinically relevant, antigens expressedby the component cell lines.

The six cell lines included in the NSCLC vaccine described herein wereselected to express a wide array of TAAs, including those known to beimportant to antitumor immunity. As a result, the number of TAAs thatcan be targeted using the exemplary six-cell line composition, and theexpression levels of the antigens, is higher than belagenpumatucel-L. Asdescribed earlier, to further enhance antigenic breadth, one cell line(LK-2) was also transduced with the genes for CT83 (SEQ ID NO: 19, SEQID NO: 20) and mesothelin (SEQ ID NO: 17, SEQ ID NO: 18), two TAAs forwhich mRNA was endogenously expressed at low levels in any of the sixcomponent cell lines.

This Example demonstrates that the reduction of TGFβ1, TGFβ2, and CD276expression with concurrent overexpression of GM-CSF, CD40L, and IL-12 inof the NSCLC vaccine comprising two cocktails, each cocktail composed ofthree cell line components, a total of 6 component cell lines,significantly increases the antigenic breadth and magnitude of cellularimmune responses compared to belagenpumatucel-L.

Reduction of TGFβ2 Secretion in the Belagenpumatucel-L Cell Lines

The cell line components of the belagenpumatucel-L cocktail, NCI-H460,NCI-H520, SK-LU-1, and Rh2 were transduced with lentiviral particlesexpressing shRNA specifically targeting TGFβ2 (SEQ ID NO: 24) andresulting TGFβ2 levels in the modified cell lines was determined asdescribed in Example 5. TGFβ2 secretion levels in the modified cellswere below the lower limit of quantification of the ELISA assay forNCI-H520 and SK-LU-1 and the MDD (42.0 μg/10⁶ cells/24 hours was used toestimate the percent reduction relative to the parental cell line.Compared to the parental, unmodified cell lines, TGFβ2 secretion wasreduced 84% in NCI-H460, 99% in NCI-H520, 84% in SK-LU-1, and 74% inRh2. Reduction of TGFβ1 and TGFβ2 for NSCLC cocktail A and cocktail Blevels are described in Table 41. The NSCLC vaccine was prepared asdescribed in Example 27.

Antigen Specific and Tumor Cell Specific IFNγ Production to NSCLCVaccine-A, NSCLC Vaccine-B, and Belagenpumatucel-L

Cellular immune responses to antigens and parental, unmodified cellswere determined by IFNγ ELISpot following autologous DC and PBMCco-culture as described in Example 8 with modifications as describedbelow.

The autologous DC and PBMC co-cultures were adjusted to model the invivo administration of the belagenpumatucel-L and the NSCLC vaccine.Belagenpumatucel-L was administered in a single site and NSCLC vaccine-Aand NSCLC vaccine-B are administered in two separate injection sites. Inthe autologous DC and PBMC co-culture representing Belagenpumatucel-L,3.75×10⁵ of NCI-H460, NCI-H520, SK-LU-1, Rh2 modified cells, 1.5×10⁶total modified cells, were co-cultured with 1.5×10⁶ iDCs. NSCLCvaccine-A, 5.00×10⁵ of the modified NCI-H460, NCI-H520, A549 cells,1.5×10⁶ total modified cells, were co-cultured with 1.5×10⁶ iDCs. ForNSCLC vaccine-B, 5.0×10⁵ of the modified DMS 53, NCI-H23, and LK-2cells, 1.5×10⁶ total modified cells, were co-cultured with 1.5×10⁶ iDCs.Following co-culture, cellular immune responses directed againstparental tumor cell lines and antigens were determined by IFNγ ELISpot.CD14-PBMCs from the Belagenpumatucel-L co-culture were stimulated inseparate wells with unmodified NCI-H460, NCI-H520, SK-LU-1, or Rh2(n=4/cell line/donor). CD14⁻ PBMCs from NSCLC vaccine-A cocktail werestimulated in separate wells with either NCI-H460, NCI-H520, or A549(n=4/cell line/donor). CD14⁻ PBMCs from NSCLC vaccine-B cocktail werestimulated in separate wells with either DMS 53, LK-2, or NCI-H23(n=4/cell line/donor). Antigen specific responses were determined usingCD14⁻ PBMCs isolated from the same belagenpumatucel-L, NSCLC vaccine-A,and NSCLC vaccine-B co-cultures (n=4/donor/antigen). IFNγ productionresponses were determined against the parental, unmodified cell linescomprising the belagenpumatucel-L vaccine, NSCLC vaccine-A and NSCLCvaccine-B and to exemplary tumor-associated antigens (TAAs),tumor-specific antigens (TSA), and cancer/testis antigens (CTA).

Reduction of TGFβ1, TGFβ2, and CD276 Expression with ConcurrentOverexpression of GM-CSF, CD40L, and IL-12 in 6 Component Cell Line, 2Cocktail Approach, Significantly Increases Cellular Immune ResponsesCompared to Reduction of TGFβ2 in a 4-Component Cell Line, SingleCocktail Immunotherapy Approach

IFNγ responses induced by the belagenpumatucel-L, Cocktail A andCocktail B, against parental tumor cells and antigens were determinedwith following co-culture of CD14-PBMCs and DCs derived from 8 healthy,HLA diverse donors. PBMCs co-cultured with DCs loaded with the modifiedbelagenpumatucel-L NCI-H460, NCI-H520, SK-LU-1, Rh2 component cell lineswere stimulated with parental, unmodified, NCI-H460, NCI-H520, SK-LU-1,Rh2 cells (n=4/donor/cell line). PBMCs co-cultured with DCs loaded withCocktail A were stimulated with parental, unmodified, NCI-H460,NCI-H520, A549 cells (n=4/donor/cell line). PBMCs co-cultured with DCsloaded with Cocktail B were stimulated with parental, unmodified, DMS53, NCI-H23, and LK-2 cells (n=4/donor/cell line). The average SFU ofthe replicates (n=4) for each donor variable is reported±SEM. The NSCLCvaccine unit dose elicited significantly more robust tumor cell specificIFNγ responses (7,613±1,763 SFU) (n=8) compared to belagenpumatucel-L(1,850±764 SFU) (n=8) (p=0.0148, Mann-Whitney U test) (FIG. 66A).Donor-specific IFNγ responses to belagenpumatucel-L, NSCLC vaccineCocktail A, NSCLC vaccine Cocktail B, and NSCLC vaccine unit dose areshown in FIG. 67A.

Table 46 shows that the distribution of IFNγ responses to Cocktail A andCocktail B varied on a per donor basis emphasizing that that increasingthe number of cell lines of cell line components and delivery sites hasthe potential to reach a boarder population than a single composition of4 cell lines.

TABLE 46 IFNγ responses Cock- Cock- NSCLC tail tail Vaccine Foldbelagenpumatucel-L A B Unit Dose Increase* Donor 1 473 943 75 1,018  2.2Donor 2 6,180 6,180 4,983 11,163  3.4 Donor 3 339 926 1,303 2,229  6.6Donor 4 4,163 4,413 829 5,242  1.3 Donor 5 1,476 3,039 8,780 11,819  8.0Donor 6 1,200 11,240 2,330 13,570 11.3 Donor 7 225 2,107 1,956 4,06318.1 Donor 8 740 7,848 3,950 11,798 15.9 Mean 1,850 4,587 3,026 7,613SEM 764 1,287 999 1,763 *Fold Increase of IFNγ SFU induced by IA UnitDose relative to belagenpumatucel-L. (n = 4/Donor)

NSCLC vaccine Cocktail A and Cocktail B also induced more robust antigenspecific IFNγ responses to an exemplary panel of antigens associatedwith NSCLC and other solid tumor indications. PBMCs co-cultured with DCsloaded with the belagenpumatucel-L, NSCLC vaccine Cocktail A, or NSCLCvaccine Cocktail B were stimulated with peptides pools containing knownantigen specific T cell epitopes for a broad range of HLA haplotypes(n=4/donor/antigen). The average SFU of the replicates for each antigenand donor (n=4) is reported±SEM in Table 47 and in FIG. 67B. The NSCLCvaccine unit dose significantly increased the mean magnitude and breadthof antigen specific IFNγ production (6,576±2,147 SFU) (n=8) relative tothe belagenpumatucel-L (392±157 SFU) in 8 Donors (p=0.0002, Mann-WhitneyU test) (FIG. 66B).

TABLE 47 Mean magnitude of antigen specific IFNγ production NSCLCVaccine Fold belagenpumatucel-L Unit Dose Increase* Donor 1 172  3,84722.4 Donor 2 125  7,493 59.9 Donor 3 23  1,248 55.4 Donor 4 35  2,50071.4 Donor 5 275  3,723 13.5 Donor 6 977 20,603 21.1 Donor 7 340  6,74819.8 Donor 8 1,191  6,447  5.4 Mean 392  6,576 SEM 157  2,147 *FoldIncrease of IFNγ SFU induced by NSCLC vaccine Unit Dose relative tobelagenpumatucel-L. (n = 4/Donor)

Example 29: Preparation of Glioblastoma Multiforme (GBM) Cancer Vaccine

This Example demonstrates that reduction of TGFβ1, TGFβ2, and CD276expression with concurrent overexpression of GM-CSF, CD40L, and IL-12 ina vaccine composition of two cocktails, each cocktail composed of threecell lines for a total of 6 cell lines, significantly increased themagnitude of cellular immune responses to at least 10 GBM-associatedantigens in an HLA-diverse population. As described herein, the firstcocktail, GBM vaccine-A, is composed of cell line LN-229 that was alsomodified to express modPSMA, cell line GB-1, and cell line SF-126 thatwas also modified to express modTERT. The second cocktail, GBMvaccine-B, is composed of cell line DBTRG-05MG, cell line KNS 60 thatwas also modified to express modMAGEA1, hCMV pp65, and EGFRvIII, andcell line DMS 53. The 6 component cell lines collectively express atleast twenty-two antigens that can provide an anti-GBM tumor response.

Identification of Glioblastoma Multiforme Vaccine Components

Initial cell line selection criteria identified seventeen vaccinecomponent cell lines for potential inclusion in the GBM vaccine.Additional selection criteria were applied to narrow the seventeencandidate cell lines to eight cell lines for further evaluation inimmunogenicity assays. These criteria included: endogenous GBMassociated antigen expression, lack of expression of additionalimmunosuppressive factors, such as IL-10 or IDO1, expression of GBMspecific CSC markers, ethnicity and age of the patient from which thecell line was derived, GBM histological and molecular subtype (whenavailable), and the methylation status of the O⁶-methylguanine-DNAmethyltransferase (MGMT) promoter (when available).

GBM tumors are enriched with a heterogenous population of CSCs thatexpress a diverse array of CSC markers (Table 2). Expression of thirteenGBM associated CSC markers, ABCG2, ALDH1A1, BMI-1, FUT4, CD44, CD49f,CD90, PROM1, CXCR4, Musashi-1, Nestin, MYC, and SOX2 by GBM tumors wasconfirmed in patient tumor sample data downloaded from the publiclyavailable database, cBioPortal (cbioportal.org) (Cerami, E. et al.Cancer Discovery. 2012; Gao, J. et al. Sci Signal. 2013.) between Feb.23, 2020 through Jul. 2, 2020 (FIG. 68C). The HUGO Gene NomenclatureCommittee (HGNC) gene symbol was included in the search and mRNAexpression was downloaded for each CSC marker.

Expression of TAAs and CSC markers by candidate component cell lines wasdetermined by RNA expression data sourced from Broad Institute CancerCell Line Encyclopedia (CCLE). The HGNC gene symbol was included in theCCLE search and mRNA expression was downloaded for each TAA or CSCmarker. Expression of a TAA or CSC marker by a cell line was consideredpositive if the RNA-seq value (FPKM) was greater than one. Eight of theseventeen GBM vaccine candidate components were identified for furtherevaluation: DBTRG-05MG, LN-229, A-172, YKG-1, U-251 MG, GB-1, KNS 60,and SF-126 based on the selection criteria described above. The eightcandidate component cell lines expressed seven to ten CSC markers (FIG.68B) and eleven to fourteen TAAs (FIG. 68A). As described herein, theCSC-like cell line DMS 53 is included as one of the 6 cell lines.

Immunogenicity of the unmodified GBM component cell line candidates wasevaluated by IFNγ ELISpot as described in Example 9 for three HLAdiverse healthy donors (n=4 per donor). Donor HLA-A and HLA-B alleleswere as follows: Donor 1, A*02:01 B*35:01 and A*31:01 B*35:03; Donor 2,A*01:01 B*30:01 and A*02:01 B*12:02, Donor 3, A*02:01 B*15:07 and A24:02B*18:01. LN-229 (5,039±637 SFU) and DBTRG-05MG (6,094±734 SFU) were moreimmunogenic than A-172 (808±152 SFU), YKG-1 (576±154), U-251 MG(2,314±434), GB-1 (908±284 SFU), KNS-60 (2,177±415 SFU) and SF-126(1,716±332 SFU). (FIG. 69A) LN-229 was selected to be included invaccine cocktail A and DBTRG-05MG was selected to be included in vaccinecocktail B as described further herein.

Immunogenicity of DBTRG-05MG and LN-229 was evaluated in eight differentcombinations of three component cell lines, four combinations containedDBTRG-05MG and four combinations contained LN-229 (FIG. 69C). IFNγresponses were determined against the three component cell lines withinin the eight potential vaccine cocktails by IFNγ ELISpot as described inExample 8 using the same three healthy donors described above(n=4/donor). IFNγ responses were detected for all eight cocktails and toeach cell line component in each cocktail. Responses to the individualcocktail component cell lines were notably decreased compared to IFNγresponses detected for single cell line components. In all eightcombinations evaluated, DBTRG-05MG and LN-229 remained the mostimmunogenic (FIG. 69B).

The cells in the vaccine described herein were selected to express awide array of TAAs, including those known to be important specificallyfor GBM antitumor responses, such as IL13Ra2, and also TAAs known to beimportant for targets for GBM and other solid tumors, such TERT. Asshown herein, to further enhance the array of TAAs, LN-229 wastransduced with a gene encoding modPSMA, SF-126 was transduced with agene encoding modTERT and KNS-60 was transduced with genes encodingmodMAGEA1, hCMV pp65, and the 14 amino sequence spanning the in-framedeletion of 267 amino acids of EGFR that results in an activatingmutated form of EGFR, EGFRvIII, as described herein.

TERT, PSMA and MAGEA1 were endogenously expressed in one of the sixcomponent cell lines, and the activating mutation EGFRvIII and GBMassociated viral antigen hCMV pp65 were not endogenously expressed inone or more cell lines at >1.0 FPKM as described below (FIG. 70).Expression of the transduced antigens modTERT (SEQ ID NO: 35; SEQ ID NO:36) by SF-126 (FIG. 71A), modPSMA (SEQ ID NO: 37; SEQ ID NO: 38) byLN-229 (FIG. 71B), modMAGEA1 (SEQ ID NO: 39; SEQ ID NO: 40) by KNS 60(FIG. 71C), EGFRvIII (SEQ ID NO: 39; SEQ ID NO: 40) by KNS 60 (FIG.71D), and hCMV pp65 (SEQ ID NO: 39; SEQ ID NO: 40) by KNS 60 (FIG. 71E),were detected by flow cytometry as described herein. Expression ofEGFRvIII and hCMV pp65 by KNS 60 were also detected by RT-PCR asdescribed herein (FIG. 71F). The genes for MAGEA1, EGFRvIII, and hCMVpp65 are encoded in the same lentiviral transfer vector separated byfurin cleavage sites. IFNγ production to the transduced antigens isdescribed herein.

Because of the need to maintain maximal heterogeneity of antigens andclonal subpopulations the comprise each cell line, the gene modifiedcell lines utilized in the present vaccine have been established usingantibiotic selection and flow cytometry and not through limitingdilution subcloning.

The mRNA expression of representative TAAs in the present vaccine areshown in FIG. 70A. The present vaccine has high expression of allidentified twenty-two commonly targeted and potentially clinicallyrelevant TAAs for inducing a GBM antitumor response. Some of these TAAsare known to be primarily enriched in GBM tumors and some can alsoinduce an immune response to GBM and other solid tumors. Expression ofthe twenty-two prioritized GBM TAAs was determined in 170 GBM patientsamples using the same methods and 170 patient samples used to confirmthe expression of GBM CSC markers described above. Eighteen of theprioritized GBM TAAs were expressed by 100% of samples, 19 TAAs wereexpressed by 97.2% of samples, 20 TAAs were expressed by 79.4% ofsamples, 21 TAAs were expressed by 32.9% of samples, and 22 TAAs wereexpressed by 1.8% samples (FIG. 70B). Based on the expression andimmunogenicity data presented herein, the cell lines identified in Table48 were selected to comprise the present GBM vaccine.

TABLE 48 Glioblastoma vaccine cell lines and histology Cocktail CellLine Name Histology A LN-229 Glioblastoma Multiforme A GB-1 GlioblastomaMultiforme A SF-126 Glioblastoma Multiforme B DBTRG-05MG GlioblastomaMultiforme B KNS-60 Glioblastoma Multiforme B DMS 53 Lung Small CellCarcinoma

CD276 Expression

The LN-229, GB-1, SF-126, KNS-60, and DMS 53 component cell linesexpressed CD276 and expression was knocked out by electroporation withZFN as described in Example 13 and elsewhere herein. DBTRG-05MG wastransduced with lentiviral particles expressing shRNA specific forknockdown of CD276 (shCD276,ccggtgctggagaaagatcaaacagctcgagctgtttgatctttctccagcatttttt (SEQ ID NO:71). Because it was desirable to maintain as much tumor heterogeneity aspossible, the electroporated and shRNA modified cells were not cloned bylimiting dilution. Instead, the cells were subjected to multiple roundsof cell sorting by FACS as described in Example 13.

Expression of CD276 was determined by extracellular staining of modifiedand parental cell lines with PE α-human CD276 (BioLegend, clone DCN.70)on Day 1 (before irradiation) and Day 3 (48 hours post-irradiation).Irradiation did not impact CD276 expression levels and Day 1 MFI valuesare reported. Unstained cells and isotype control PE α-mouse IgG1(BioLegend, clone MOPC-21) stained parental and CD276 KO cells served ascontrols. The MFI of the isotype control was subtracted from reportedvalues for both the parental and modified cell lines. Percent reductionof CD276 expression is expressed as: (1−(MFI of the CD276KO cellline/MFI of the parental))×100). MFI is normalized to 100,000 cells.Reduction of CD276 expression is described in Table 49. These data showthat gene editing of CD276 with shRNA or ZFN resulted in greater than58.5% CD276-negative cells in all six vaccine component cell lines.

TABLE 49 Reduction of CD276 expression Parental Cell Modified Cell %Reduction Cell line Line MFI Line MFI CD276 LN-229 17,549 176 99.0 GB-131,439 137 99.6 SF-126 25,608 18 99.9 DBTRG-05MG 67,196 27,879 58.5KNS-60 12,218 122 99.0 DMS 53 11,928 24 99.8 MFI reported with isotypecontrols subtracted

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12

Cell lines were X-ray irradiated at 100 Gy prior to plating in 6-wellplates at 2 cell densities (5.0e5 and 7.5e5) in duplicate. The followingday, cells were washed with PBS and the media was changed to SecretionAssay Media (Base Media+5% CTS). After 48 hours, media was collected forELISAs. The number of cells per well was counted using the Luna cellcounter (Logos Biosystems). Total cell count and viable cell count wererecorded. The secretion of cytokines in the media, as determined byELISA, was normalized to the total cell count recorded.

TGFβ1 secretion was determined by ELISA according to manufacturersinstructions (Human TGFβ1 Quantikine ELISA, R&D Systems #SB100B). Fourdilutions were plated in duplicate for each supernatant sample. If theresults of the ELISA assay were below the LLD, the percentage decreaserelative to parental cell lines was estimated by the number of cellsrecovered from the assay and the lower limit of detection, 15.4 μg/mL.If TGFβ1 was detected in >2 samples or dilutions the average of thepositive values was reported with the n of samples run.

TGFβ2 secretion was determined by ELISA according to manufacturersinstructions (Human TGFβ2 Quantikine ELISA, R&D Systems #SB250). Fourdilutions were plated in duplicate for each supernatant sample. If theresults of the ELISA assay were below the LLD, the percentage decreaserelative to parental cell lines was estimated by the number of cellsrecovered from the assay and the lower limit of detection, 7.0 μg/mL. IfTGFβ2 was detected in >2 samples or dilutions the average of thepositive values was reported with the n of samples run.

GM-CSF secretion was determined by ELISA according to manufacturersinstructions (GM-CSF Quantikine ELISA, R&D Systems #SGM00). Fourdilutions were plated in duplicate for each supernatant sample. If theresults of the ELISA assay were below the LLD, the percentage increaserelative to parental cell lines was estimated by the number of cellsrecovered from the assay and the lower limit of detection, 3.0 μg/mL. IfGM-CSF was detected in >2 samples or dilutions the average of thepositive values was reported with the n of samples run.

IL-12 secretion was determined by ELISA according to manufacturer'sinstructions (LEGEND MAX Human IL-12 (p70) ELISA, Biolegend #431707).Four dilutions were plated in duplicate for each supernatant sample. Ifthe results of the ELISA assay were below the LLD, the percentageincrease was estimated by the number of cells recovered from the assayand the lower limit of detection, 1.2 μg/mL. If IL-12 was detected in >2samples or dilutions the average of the positive values was reportedwith the n of samples run.

shRNA Downregulates TGF-β Secretion

Following CD276 knockout, TGFβ1 and TGFβ2 secretion levels were reducedusing shRNA and resulting levels determined as described above. Of theparental cell lines in GBM vaccine-A, LN-229, GB-1 and SF-126 secretedmeasurable levels of TGFβ1 and TGFβ2. Of the parental cell lines in GBMvaccine-B, DBTRG-05MG, KNS 60, and DMS 53 secreted measurable levels ofTGFβ1 and TGFβ2. Reduction of TGFβ2 secretion by the DMS 53 cell line isdescribed in Example 5 and resulting levels determined as describedabove.

The five component cell lines of GBM origin were transduced with TGFβ1shRNA to decrease secretion of TGFβ1. The lentiviral particles encodingTGFβ1 shRNA also encoded the gene for expression of membrane bound CD40Lunder the control of a different promoter. This allowed for simultaneousreduction of TGFβ1 and expression of membrane bound CD40L. SF-126 andKNS 60 were subsequently transduced with lentiviral particles encodingTGFβ2 shRNA and GM-CSF (SEQ ID NO: 6). This allowed for simultaneousreduction of TGFβ2 and expression of GM-CSF in both cell lines.

DBTRG-05MG and GB-1 were gene modified with only TGFβ1 shRNA. TGFβ1 andTGFβ2 promote cell proliferation and survival. In some cell lines, as insome tumors, reduction of TGFβ signaling can induce growth arrest andlead to cell death. In neuronal cells, such as GBM, loss of TGFβsignaling is also associated with cell death. TGFβ1 knockdown wasselected for modification because it is considered a more potentimmunosuppressive factor relative to TGFβ2 and retaining some TGFβsignaling is likely necessary for proliferation and survival of thesecell lines. LN-229 secreted TGFβ2 at a detectable, but low, level andwas not modified with TGFβ2 shRNA. These cells are described by theclonal designation DK2. As described in Example 26, DMS 53 was modifiedwith shRNA to reduce secretion of TGFβ2 and not TGFβ1. These cells aredescribed by the clonal designation DK4. The remaining cell lines weredouble modified with TGFβ1 shRNA and TGFβ2 shRNA. These cells aredescribed by the clonal designation DK6.

Table 50 shows the percent reduction in TGFβ1 and/or TGFβ2 secretion ingene modified component cell lines compared to unmodified, parental,cell lines. Gene modification resulted in 49% to 80% reduction of TGFβ1secretion. Gene modification of TGFβ2 resulted in 51% to 99% reductionin secretion of TGFβ2. TGFβ1 shRNA modified DBTRG-05MG secreted lessTGFβ2 than the unmodified, parental cell line. Lower secretion of TGFβ2by the modified cell line was confirmed in multiple independentexperiments. Lower secretion of TGFβ2 following TGFβ1 knockdown was notobserved in other component cell lines.

TABLE 50 TGF-β Secretion (pg/10⁶ cells/24 hr) in Component Cell LinesCell Line Cocktail Clone TGFβ1 TGFβ2 LN-229 A Wild type 1,874 ± 294  116 ± 19  LN-229 A DK2 384 ± 47  73 ± 41 LN-229 A Percent reduction 80%NA GB-1 A Wild type 204 ± 28  481 ± 51  GB-1 A DK2 66 ± 16 438 ± 59 GB-1 A Percent reduction 68% NA SF-126 A Wild type 2,818 ± 258   784 ±98  SF-126 A DK6 792 ± 188 * ≤11 SF-126 A Percent reduction 72% 99%DBTRG-05MG B Wild type 6,626 ± 389   2,664 ± 461   DBTRG-05MG B DK23,365 ± 653   612 ± 190 DBTRG-05MG B Percent reduction 49% NA KNS 60 BWild type 3,308 ± 615   1,451 ± 235   KNS 60 B DK6 1,296 ± 110   36 ± 11KNS 60 B Percent reduction 61% 97% DMS 53 B Wild type 106 ± 10  486 ±35  DMS 53 B DK4 219 ± 33  238 ± 40  DMS 53 B Percent reduction NA 51%DK6: TGFβ1/TGFβ2 double knockdown; DK4: TGFβ2 single knockdown; DK2:TGFβ1 single knockdown; * = estimated using LLD, not detected; NA = notapplicable

Based on a dose of 5×10⁵ of each component cell line, the total TGFβ1and TGFβ2 secretion by the modified GBM vaccine-A and GBM vaccine-B andrespective unmodified parental cell lines are shown in Table 51. Thesecretion of TGFβ1 by GBM vaccine-A was reduced by 75% and TGFβ2 by 62%pg/dose/24 hr. The secretion of TGFβ1 by GBM vaccine-B was reduced by51% and TGFβ2 by 74% pg/dose/24 hr.

TABLE 51 Total TGF-β Secretion (pg/dose/24 hr) in GBM vaccine-A and GBMvaccine-B Cocktail Clones TGFβ1 TGFβ2 A Wild type 2,448   691 DK2/6  621   261 Percent reduction 75% 62% B Wild type 5,020 2,301 DK2/4/62,440   600 Percent reduction 51% 74%

GM-CSF Secretion

Two GBM component cell lines, KNS 60 and SF-126, were transduced withlentiviral particles containing both TGFβ2 shRNA and the gene to expressGM-CSF (SEQ ID NO: 6) under the control of a different promoter. Thisallowed for simultaneous reduction of TGFβ2 secretion and expression ofGM-CSF. The DBTRG-05MG, LN-229 and GB-1 cell lines were transduced withlentiviral particles to only express GM-CSF (SEQ ID NO: 7). DMS 53 wasmodified to secrete GM-CSF as described in Example 24 and elsewhereherein. The results are shown in Table 52 and described below.

Secretion of GM-CSF increased at least 19,000-fold in all modifiedcomponent cell lines compared to unmodified, parental cell lines. In GBMvaccine-A component cell lines, secretion of GM-CSF increased303,000-fold by LN-229 compared to the parental cell line (≤0.002 ng/10⁶cells/24 hr), 409,000-fold by GB-1 compared to the parental cell line(≤0.001 ng/10⁶ cells/24 hr), and 19,000-fold by SF-126 compared to theparental cell line (≤0.003 ng/10⁶ cells/24 hr). In GBM vaccine-Bcomponent cell lines secretion of GM-CSF increased 1,209,500-fold byDBTRG-05MG compared to the parental cell line (≤0.002 ng/10⁶ cells/24hr), 109,667-fold by KNS 60 compared to the parental cell line (≤0.003ng/10⁶ cells/24 hr) and 39,450-fold by DMS 53 compared to the parentalcell line (≤0.004 ng/10⁶ cells/24 hr).

TABLE 52 GM-CSF Secretion in ComponentCell Lines GM-CSF GM-CSF Cell Line(ng/10⁶ cells/24 hr) (ng/dose/24 hr) LN-229 606 ± 228 303 GB-1 409 ± 161205 SF-126 57 ± 13 29 Cocktail A Total 1,072 537 DBTRG-05MG 2,419 ±721   1,210 KNS 60 329 ± 45  165 DMS 53 158 ± 15  79 Cocktail B Total2,906 1,454

Based on a dose of 5×10⁵ of each component cell line, the total GM-CSFsecretion for GBM vaccine-A was 537 ng per dose per 24 hours. The totalGM-CSF secretion for GBM vaccine-B was 1,454 ng per dose per 24 hours.The total GM-CSF secretion per dose was therefore 1,991 ng per 24 hours.

Membrane Bound CD40L (CD154) Expression

The component cell lines were transduced with lentiviral particles toexpress membrane bound CD40L vector as described above. The methods todetect expression of CD40L by the five GBM cell line components aredescribed herein. The methods used to modify DMS 53 to express CD40L aredescribed in Example 15. Evaluation of membrane bound CD40L by all sixvaccine component cell lines is described below.

CD40L expression was evaluated by flow cytometry with an anti-CD40Lmonoclonal antibody as described in Example 15. CD40L expression wasdetermined on Day 1 (pre-irradiation) and Day 3 (post-irradiation).Irradiation did not impact expression levels and Day 1 CD40L expressionis reported. If subtraction of the MFI of the isotype control resultedin a negative value, an MFI of 1.0 was used to calculate the foldincrease in expression of CD40L by the modified component cell linerelative to the unmodified cell line. The results shown in FIG. 72 anddescribed below demonstrate CD40L membrane expression was substantiallyincreased in all six cell GBM vaccine component cell lines.

FIG. 72 shows the expression of membrane bound CD40L by the GBM vaccinecomponent cell lines. Expression of membrane bound CD40L increased atleast 172-fold in all component cell lines compared to unmodified,parental cell lines. In GBM vaccine-A component cell lines, expressionof CD40L increased 11,628-fold by LN-229 (11,628 MFI) compared to theparental cell line (0 MFI), 233-fold by GB-1 (4,464 MFI) compared to theparental cell line (19 MFI), and 172-fold by SF-126 (5,526) compared tothe parental cell line (32 MFI). In GBM vaccine-B component cell linesexpression of CD40L increased 20,510-fold by DBTRG-05MG compared to theparental cell line (0 MFI), 5,599-fold by KNS 60 compared to theparental cell line (0 MFI), and 88,261-fold by DMS 53 compared to theparental cell line (0 MFI).

IL-12 Expression

The component cell lines were transduced with the IL-12 vector asdescribed in Example 17 and resulting IL-12 p70 expression determined asdescribed above and herein. The results are shown in Table 53 anddescribed below.

Secretion of IL-12 increased at least 45,000-fold in all component celllines modified to secrete IL-12 p70 compared to unmodified, parentalcell lines. In GBM vaccine-A component cell lines, secretion of IL-12increased 81,000-fold by LN-229 compared to the parental cell line(≤0.001 ng/10⁶ cells/24 hr), 50,000-fold by GB-1 compared to theparental cell line (≤0.0002 ng/10⁶ cells/24 hr), and 45,000-fold bySF-126 compared to the parental cell line (≤0.001 ng/10⁶ cells/24 hr).In GBM vaccine-B component cell lines expression of IL-12 increased133,560-fold by DBTRG-05MG compared to the parental cell line (≤0.001ng/10⁶ cells/24 hr) and 116,000-fold by KNS 60 compared to the parentalcell line (≤0.001 ng/10⁶ cells/24 hr). DMS 53 was not modified tosecrete IL-12.

TABLE 53 IL-12 secretion in component cell lines IL-12 IL-12 Cell Line(ng/10⁶ cells/24 hr) (ng/dose/24 hr) LN-229 81 ± 4 41 GB-1 10 ± 1 5SF-126 45 ± 7 23 Cocktail A Total 136 69 DBTRG-05MG 134 ± 24 67 KNS 60116 ± 5  58 DMS 53 NA NA Cocktail B Total 250 125

Based on a dose of 5×10⁵ of each component cell line, the total IL-12secretion for GBM vaccine-A was 69 ng per dose per 24 hours. The totalIL-12 secretion for GBM vaccine-B was 125 ng per dose per 24 hours. Thetotal IL-12 secretion per dose was therefore 194 ng per 24 hours.

Stable Expression of modPSMA by the LN-229 Cell Line

As described above, the cells in the vaccine described herein wereselected to express a wide array of TAAs, including those known to beimportant to GBM antitumor immunity. To further enhance the array ofantigens, the LN-229 cell line that was modified to reduce the secretionof TGFβ1, reduce the expression of CD276, and to express GM-CSF,membrane bound CD40L, and IL-12 was also transduced with lentiviralparticles expressing the modPSMA antigen (SEQ ID NO: 37, SEQ ID NO: 38).

The expression of modPSMA was characterized by flow cytometry.Unmodified parental and modified cells were stained intracellular with0.06 μg/test anti-mouse IgG1 anti-PSMA antibody (AbCam ab268061, CloneFOLH1/3734) followed by 0.125 ug/test AF647-conjugated goat anti-mouseIgG1 antibody (Biolegend #405322). The MFI of the isotype controlstained parental and modified cells was subtracted from the MFI of cellsstained anti-PSMA. MFI was normalized to 100,000 events. Fold increasein antigen expression was calculated as: (background subtracted modifiedMFI/background subtracted parental MFI). Expression of PSMA increased inthe modified cell line (533,577 MFI) 38-fold over that of the parentalcell line (14,008 MFI) (FIG. 71B).

Stable Expression of modMAGEA1, EGFRvIII, hCMV-Pp65 by the KNS 60 CellLine

As described above, the cells in the vaccine described herein wereselected to express a wide array of TAAs, including those known to beimportant to antitumor immunity. To further enhance the array ofantigens, the KNS 60 cell line that was modified to reduce the secretionof TGFβ1 and TGFβ2, reduce the expression of CD276, and to expressGM-CSF, membrane bound CD40L and IL-12 was also transduced withlentiviral particles expressing the modMAGEA1, hCMV pp65, and EGFRvIIIantigens. The modMAGEA1, hCMV pp65, and EGFRvIII antigens are linked bya furin cleavage site (SEQ ID NO: 39, SEQ ID NO: 40).

The expression of modMAGEA1, hCMV pp65, and EGFRvIII was characterizedby flow cytometry. Unmodified parental and modified cells were stainedintracellular to detect the expression of each antigen as follows. Forthe detection of modMAGEA1, cells were first stained with mouse IgG1anti-MAGEA1 antibody (SC-71539, Clone 3F256) (0.03 ug/test) followed byAF647-conjugated goat anti-mouse IgG1 antibody (Biolegend #405322)(0.125 ug/test). For the detection of hCMVpp65, cells were first stainedwith mouse IgG1 anti-pp65 antibody (AbCam ab31624, Clone 1-L-11) (0.06ug/test) followed by AF647-conjugated goat anti-mouse IgG1 antibody(Biolegend #405322) (0.125 ug/test). For the detection of EGFRvIII,cells were first stained with mouse IgG1 anti-EGFRvIII antibody (NovusNBP2-50599, Clone DH8.3) (0.06 ug/test) followed by AF647-conjugatedgoat anti-mouse IgG1 antibody (Biolegend #405322) (0.125 ug/test). TheMFI of the isotype control stained cells was subtracted from the MFI ofthe cells stained for MAGEA1, hCMV pp65, or EGFRvIII. MFI was normalizedto 100,000 events. Fold increase in antigen expression was calculatedas: (background subtracted modified MFI/background subtracted parentalMFI).

Expression of hCMV pp65 and EGFRvIII was also confirmed by RT-PCR (FIG.81F). 1.0-3.0×10⁶ cell were used for RNA isolation. RNA was isolatedusing Direct-zol™ RNA MiniPrep kit (ZYMO RESEARCH, catalog number:R2051) per the manufacturers instructions. RNA quantification wasperformed using NanoDrop™ OneC (Thermo Scientific™ catalogue number13-400-519). For reverse transcription, 1 μg of RNA was reversetranscribed using qScript cDNA SuperMix (Quantabio, catalogue number:95048-025) per the manufacturer's instructions to cDNA. After completionof cDNA synthesis, the reaction was diluted two times and 2 μL of cDNAwere used for amplification. For hCMV pp65, the forward primer designedto anneal at the 1925-1945 base pair (bp) location in the transgene(CGGACTGCTGTGTCCTAAGAG (SEQ ID NO: 118)) and reverse primer designed toanneal at 2414-2435 bp location in the transgene (GCTGTCCTCGTCTGTATCTTCC(SEQ ID NO: 119)) and yield 511 bp product. For EGFRvIII, the forwardprimer was designed to anneal at the 839-858 bp location in thetransgene (TGTGAAGGTGCTGGAATACG (SEQ ID NO: 120)) and reverse primerdesigned to anneal at the 1252-1271 bp location in the transgene(GCCGGTAAAGTAGGTGTGCT (SEQ ID NO: 121)) and yield 433 bp product.β-tubulin primers that anneal to variant 1, exon 1 (TGTCTAGGGGAAGGGTGTGG(SEQ ID NO: 122) and exon 4 (TGCCCCAGACTGACCAAATAC (SEQ ID NO: 123))were used as a control. PCR to detect hCMV pp65, EGFRvIII and β-tubulinwas completed as follows: initial denaturation, 98° C. for 30 seconds,followed by 25 cycles of denaturation at 98° C. for 5 to 10 seconds,annealing at 58° C. for 10 to 30 seconds, and extension at 72° C. for 30seconds. After the 25 cycles final extension of 2 min at 72° C. wascompleted and the reaction held at 10° C. until detection of the PCRproducts by gel electrophoresis. After completion of PCR, Lel LoadingDye, Purple (6×) (New England BioLabs, #B70245) was added at a 1×concentration. The PCR product was then run a 2% agarose gel (LonzaSeaKem® LE Agarose, #50004) along with 8 μL of exACT Gene 100 bp ladder(Fisher BioReagents, #BP2573100) for band size estimation. After thebands were appropriately separated, the gels were imaged using ChemiDocImaging System (BioRAD, #17001401). For relative quantification withβ-tubulin gene, Image Lab Software v6.0 (BioRAD) was used.

Expression of modMAGEA1 increased in the modified cell line (140,342MFI) 41-fold over that of the parental cell line (3,460 MFI) (FIG. 71C).Expression of hCMV pp65 by the modified cell line (9,545 MFI) increased9,545-fold over the that of the parental cell line (0 MFI). Subtractionof the MFI of the isotype control from the MFI of the pp65 stainedparental KNS 60 resulted in negative value. The fold increase of pp65expression in the modified cell line was calculated using 1 MFI (FIG.71E). Expression of EGFRvIII by the modified cell line (4,925 MFI)increased 5-fold over the that of the parental cell line (1,053 MFI)(FIG. 71D).

Stable Expression of modTERT by the SF-126 Cell Line

As described above, the cells in the vaccine described herein wereselected to express a wide array of TAAs, including those known to beimportant to antitumor immunity. To further enhance the array ofantigens, the SF-126 cell line that was modified to reduce the secretionof TGFβ1 and TGFβ2, reduce the expression of CD276, and to expressGM-CSF, membrane bound CD40L and IL-12 was also transduced withlentiviral particles expressing the modTERT antigen (SEQ ID NO: 35, SEQID NO: 36).

The expression of modTERT was characterized by flow cytometry.Unmodified parental and modified cells were stained intracellular withanti-rabbit IgG1 anti-TERT antibody (AbCam ab32020, Clone Y182) (0.03μg/test) followed by AF647-conjugated donkey anti-rabbit IgG1 antibody(Biolegend #406414) (0.125 ug/test). MFI was normalized to 100,000events. The MFI of the isotype control stained parental and modifiedcells was subtracted from the MFI of cells stained for parental andmodified cells. Fold increase in antigen expression was calculated as:(background subtracted modified MFI/background subtracted parental MFI).Expression of modTERT increased in the modified cell line (281,904 MFI)27-fold over that of the parental cell line (10,578 MFI) (FIG. 71A).

Immune Responses to MAGEA1, EGFRvIII, and hCMV Pp65 in GBM-Vaccine B

IFNγ responses to the MAGEA1, EGFRvIII, and hCMV pp65 antigens wereevaluated in the context of the GBM-vaccine B. Specifically, 5×10⁵ ofthe modified DMS 53, DBTRG-05MG and KNS 60 cell lines, a total of1.5×10⁶ total modified cells, were co-cultured with 1.5×10⁶ iDCs fromeight HLA diverse donors (n=4/donor). The HLA-A, HLA-B, and HLA-Calleles for each of the eight donors are shown in Table 54. The abilityto generate and immune responses in MHC Class I diverse donorsdemonstrates the GBM vaccine is has the potential to elicit CD8+ T cellresponses in a diverse patient population and is not class restricted toa specific MHC allele. CD14-PBMCs were isolated from co-culture with DCson day 6 and stimulated with peptide pools, 15-mers overlapping by 11amino acids or 15-mers overlapping by 9 amino acids, spanning the nativeprotein sequences, in the IFNγ ELISpot assay for 24 hours prior todetection of IFNγ producing cells. Peptides were sourced as follows:EGFRvIII, 15-mers overlapping by 9 amino acids, were purchased fromThermo Scientific Custom Peptide Service, MAGE A1 (JPT, PM-MAGEA1) andhCMV pp65 (JPT, PM-PP65-1). IFNγ responses to MAGEA1 significantlyincreased with the modified GBM vaccine-B (1,323±442 SFU) compared tothe unmodified GBM vaccine-B (225±64 SFU) (p=0.005, Mann-Whitney U test)(n=8) (FIG. 71I). EGFRvIII specific IFNγ responses significantlyincreased with the modified GBM vaccine-B (855±231 SFU) comparedunmodified GBM vaccine-B (165±93 SFU) (p=0.049, Mann-Whitney U test)(FIG. 71J). hCMV pp65 specific IFNγ responses significantly increasedwith modified GBM vaccine-B (5,283±1,434 SFU) compared to the unmodifiedGBM vaccine-B (814±229 SFU) (p=0.001, Mann-Whitney U test) (FIG. 71K).

Immune Responses to PSMA and TERT in GBM-Vaccine A

IFNγ responses to the PSMA and TERT were evaluated in the context ofGBM-vaccine A. Specifically, 5×10⁵ of the modified LN-229, GB-landSF-126 cell lines, a total of 1.5×10⁶ modified cells, were co-culturedwith 1.5×10⁶ iDCs from 8 HLA diverse donors (n=4/donor) (Table 54) andIFNγ responses determined by ELISpot as described above. PSMA peptides,15-mers overlapping by 9 amino acids spanning the length of the nativeantigen, were purchased from Thermo Scientific Custom Peptide Service.TERT peptides cover the full-length native antigen were purchased fromJPT (PM-TERT). TERT specific IFNγ responses with were significantlyincreased with the modified GBM vaccine-A (1,284±258 SFU) compared tothe parental, unmodified GBM vaccine-A (231±102 SFU) (p=0.011,Mann-Whitney U test) (n=8) (FIG. 71G). PSMA specific IFNγ responses withthe were significantly increased with the modified GBM vaccine-A(1,210±348 SFU) compared to the parental, unmodified GBM vaccine-A(154±22 SFU) (p=0.028, Mann-Whitney U test) (n=8) (FIG. 71H).

TABLE 54 Healthy Donor MHC-I characteristics Donor # HLA-A HLA-B HLA-C 1*02:01*03:01 *18:01 *38:01 *07:01 *12:03 2 *03:01 *25:01 *07:02 *18:01*07:02 *12:03 3 *02:01 *24:02 *08:01 *44:02 *05:01 *07:01 4 *02:01*03:01 *08:01 *51:01 *07:01 *14:02 5 *02:05 *31:01 *27:25 *50:01 *07:01*07:02 6 *23:01 *24:02 *35:03 *55:01 *27:25 *50:01 7 *30:02 *30:04*15:10 *58:02 *03:04 *06:02 8 *03:01 *32:01 *07:02 *15:17 *07:01 *07:02

Cocktails Induce Immune Responses Against Relevant TAAs

The ability of the individual component cell lines and the two GBMvaccine cocktails to induce IFNγ production against relevant GBMantigens was measured by ELISpot. PBMCs from eight HLA-diverse healthydonors (Table 54) were co-cultured with the GBM-A or GBM-B cocktails for6 days prior to stimulation with autologous DCs loaded with TAA-specificspecific peptide pools containing known MHC-I restricted epitopes.Peptides for stimulation of CD14-PBMCs were sourced as follows. Custompeptide libraries of 15-mers overlapping by 9 amino acids were orderedfrom Pierce for PSMA, WT1 and EGFRvIII. Additional 15-mer overlapping by11 amino acid peptide pools were sourced as follows: TERT (JPT,PM-TERT), MAGE A1 (JPT, PM-MAGEA1), Survivin (thinkpeptides,7769_001-011), WT1 (HER2 (JPT, PM-ERB_ECD), STEAP (PM-STEAP1), MUC1(JPT, PM-MUC1), and hCMV pp65 (JPT, PM-PP65-1). Cells were then assayedfor IFNγ secretion in the IFNγ ELISpot assay.

Approximately 60-70% of developed nations populations are hCMV positive(Hyun et al. Front. Immunol. 2017) and the hCMV status of the healthydonors in unknown. It is possible that the hCMV pp65 antigen in the GBMvaccine boosted a preexisting memory response in healthy donor PBMCs anddid not prime a de novo response. For this reason, responses to hCMV areshown separately from the other nine prioritized TAAs and are notincluded in the TAA responses illustrated in FIG. 73, FIG. 74 or Table55. Responses to the hCMV pp65 antigen in donor PBMCs when stimulatedwith parental controls or the GBM vaccine are shown in FIG. 71J. IFNγresponses to pp65 significantly increased with the GBM vaccine in sevenof eight donors compared to parental controls. Specifically, expressionof hCMV pp65 by KNS 60 significantly increased pp65 specific IFNγresponses in the context of the modified GBM vaccine-A (5,283±1,434 SFU)compared to the parental, unmodified GBM vaccine-A (814±229 SFU)(p=0.001, Mann-Whitney U test).

FIG. 73 demonstrates the GBM vaccine is capable of inducing antigenspecific IFNγ responses in eight HLA-diverse donors that aresignificantly more robust (17,316±4,171 SFU) compared to the unmodifiedparental controls (2,769±691 SFU) (p=0.004, Mann-Whitney U test) (n=8)(FIG. 73A). GBM vaccine-A and GBM vaccine-B independently demonstratedantigen specific responses significantly greater compared to parentalcontrols. Specifically, GBM vaccine-A elicited 7,716±2,308 SFU comparedto the unmodified controls (1,718±556 SFU) (p=0.038, Mann-Whitney Utest) (FIG. 73B). For GBM vaccine-A, excluding hCMV (n=9 antigens), onedonor responded to four, three donors responded to seven antigens, onedonor responded to eight antigens, and three donors responded to nineantigens. GBM vaccine-B elicited 9,601±2,413 SFU compared to parentalcontrols (1,051±365 SFU) (p<0.001, Mann-Whitney U test) (FIG. 73C). ForGBM vaccine-B, excluding hCMV (n=9 antigens), two donors responded toseven antigens, three donors responded to eight antigens, and threedonors responded to nine antigens. The GBM vaccine (vaccine-A andvaccine-B) induced IFNγ production to all nine non-viral antigens inseven of eight donors (FIG. 74) (Table 55).

TABLE 55 IFNγ Responses to unmodified and modified GBM vaccinecomponents Unmodified (SFU ± SEM) Modified (SFU ± SEM) Donor GBM GBM GBMGBM (n = 4) vaccine-A vaccine-B GBM Vaccine vaccine-A vaccine-B GBMVaccine 1 89 ± 73 738 ± 401 826 ± 469 8,653 ± 4,964 11,450 ± 6,712 20,103 ± 11,633 2 246 ± 75  594 ± 58  840 ± 112 888 ± 383 1,086 ± 642  1,974 ± 956   3 5,204 ± 1,111 433 ± 145 5,636 ± 669 ± 634 3,535 ± 2,1464,234 ± 2,748 4 1,877 ± 1,002 450 ± 317 2,327 ± 5,314 ± 3,529 20,347 ±9856   25,661 ± 13,310 5 1,295 ± 732   1,268 ± 433   2,563 6,005 ± 2,3308,130 ± 2,423 14,135 ± 4,605  6 2,330 ± 677   3,525 ± 330   5,858 ±15,253 ± 4,183  7,795 ± 2,324 23,048 ± 5,931  7 1,103 ± 503   751 ± 2231,638 ± 5,710 ± 4,657 5,965 ± 4,267 11,675 ± 8,893  8 1,600 ± 863   751± 223 2,351 ± 19,204 ± 6,757  18,497 ± 5,934  37,701 ± 12,442

Based on the disclosure and data provided herein, a whole cell vaccinefor Glioblastoma Multiforme comprising the six cancer cell lines,sourced from ATCC or JCRB, LN-229 (ATCC, CRL-2611), GB-1 (JCRB,IF050489), SF-126 (JCRB, IF050286), DBTRG-05MG (ATCC, CRL-2020), KNS 60(JCRB, IF050357) and DMS 53 (ATCC, CRL-2062) is shown in Table 56. Thecell lines represent five glioblastoma cell lines and one small celllung cancer (SCLC) cell line (DMS 53, ATCC CRL-2062). The cell lineshave been divided into two groupings: vaccine-A and vaccine-B. Vaccine-Ais designed to be administered intradermally in the upper arm andvaccine-B is designed to be administered intradermally in the thigh.Vaccine A and B together comprise a unit dose of cancer vaccine.

TABLE 56 Cell line nomenclature and modifications CD276 Cocktail CellLine TGFβ1 KD TGFβ2 KD KO/KD GM-CSF CD40L IL-12 TAA(s) A LN-229 X ND X XX X X A GB-1* X ND X X X X ND A SF-126 X X X X X X X B DBTRG- X ND  X^(∧) X X X ND 05MG* B KNS 60 X X X X X X X B DMS 53* ND X X X X X NDND = Not done. ^(∧)CD276 KD. *Cell lines identified as CSC-like cells.

Where indicated in the above table, the genes for the immunosuppressivefactors transforming growth factor-beta 1 (TGFβ1) and transforminggrowth factor-beta 2 (TGFβ2) have been knocked down using shRNAtransduction with a lentiviral vector. The gene for CD276 has beenknocked out by electroporation using zinc-finger nuclease (ZFN) orknocked down using shRNA transduction with a lentiviral vector. Thegenes for granulocyte macrophage—colony stimulating factor (GM-CSF),IL-12, CD40L, modPSMA (LN-229), modTERT (SF-126), modMAGEA1 (KNS 60),EGFRvIII (KNS 60) and hCMV pp65 (KNS 60) have been added by lentiviralvector transduction.

Example 30: Preparation of Colorectal Cancer (CRC) Vaccine

This Example demonstrates that reduction of TGFβ1, TGFβ2, and CD276expression with concurrent overexpression of GM-CSF, CD40L, and IL-12 ina vaccine composition of two cocktails, each cocktail composed of threecell lines for a total of 6 cell lines, significantly increased themagnitude of cellular immune responses to at least 10 CRC-associatedantigens in an HLA-diverse population. As described herein, the firstcocktail, CRC vaccine-A, is composed of cell line HCT-15, cell lineHuTu-80 that was also modified to express modPSMA, and cell line LS411N.The second cocktail, CRC vaccine-B, is composed of cell line HCT-116that was also modified to express modTBXT, modWT1, and the KRASmutations G12D and G12V, cell line RKO, and cell line DMS 53. The sixcomponent cell lines collectively express at least twenty antigens thatcan provide an anti-CRC tumor response.

Identification of Colorectal Vaccine Components

Sixteen vaccine component cell lines were identified using initial cellline selection criteria for potential inclusion in the CRC vaccine.Additional selection criteria were applied to narrow the sixteencandidate cell lines to eight cell lines for further evaluation inimmunogenicity assays. These criteria included: endogenous CRCassociated antigen expression, lack of expression of additionalimmunosuppressive factors, such as IL-10 or IDO1, expression ofCRC-associated CSC markers ALDH1, c-myc, CD44, CD133, Nanog, Musashi-1,EpCAM, Lgr-5 and SALL4, ethnicity and age of the patient from which thecell line was derived, microsatellite instability and CRC histologicalsubtype.

CSCs play a critical role in the metastasis and relapse of colorectalcancer (Table 2). Expression of nine CRC-associated CSC markers, by CRCtumors was confirmed in patient tumor sample data downloaded from thepublicly available database, cBioPortal (cbioportal.org) (Cerami, E. etal. Cancer Discovery. 2012; Gao, J. et al. Sci Signal. 2013) betweenOct. 1, 2019 through Oct. 20, 2020 (FIG. 75C). The HUGO GeneNomenclature Committee (HGNC) gene symbol was included in the search andRSEM normalized mRNA abundance was downloaded for each CSC marker. Of1,534 CRC patient samples 592 samples had mRNA expression data availablefor the ten CSC markers described above. A sample was consideredpositive for expression of a CRC CSC marker if Log₁₀ (RSEM+1)>0. Withinthe 592 samples 0.8% expressed 8 CSC markers (n=5), 43.9% expressed 9CSC markers (n=260) and 55.2% expressed 10 CSC markers.

Expression of TAAs and CSC markers by candidate component cell lines wasdetermined by RNA expression data sourced from Broad Institute CancerCell Line Encyclopedia (CCLE). The HGNC gene symbol was included in theCCLE search and mRNA expression was downloaded for each TAA or CSCmarker. Expression of a TAA or CSC marker by a cell line was consideredpositive if the RNA-seq value (FPKM) was greater than one. Nine of thesixteen CRC vaccine candidate components were identified for furtherevaluation: HCT-15, SW1463, RKO, HuTu80, HCT-116, LoVo, T84, LS411N andSW48 based on the selection criteria described above. The nine candidatecomponent cell lines expressed four to eight CSC markers (FIG. 75B) andseven to twelve TAAs (FIG. 75A). As described herein, the CSC-like cellline DMS 53 is included as one of the 6 cell lines and expressed fifteenCRC TAAs.

Immunogenicity of the unmodified CRC component cell line candidates wasevaluated by IFNγ ELISpot as described in Example 9 for two HLA diversehealthy donors (n=4 per donor). HLA-A and HLA-B alleles for Donor 1 wereA*02:01 B*40:01 and A*30:01 B*57:01. HLA-A and HLA-B alleles for Donor 2were A*24:02 B*18:01 and A*02:01 B*15:07. HCT-15 (2,375±774 SFU) andLoVo (1,758±311 SFU) were more immunogenic than SW1463 (170±90 SFU), RKO(280±102), HuTu80 (80±47), HCT-116 (981±433 SFU), T84 (406±185 SFU),LS411N (496±213) and SW48 (636±289 SFU)(FIG. 76A). HCT-15 and LoVo wereselected to be included in vaccine cocktail A or vaccine cocktail B asdescribed further herein.

Immunogenicity of HCT-15 and LoVo was evaluated in eight differentcombinations of three component cell lines, four combinations containedHCT-15 and four combinations contained LoVo (FIG. 76C). IFNγ responseswere determined against the three component cell lines within in theeight potential vaccine cocktails by IFNγ ELISpot as described inExample 8 using the same two donors described above (n=4/donor). IFNγresponses were detected for all eight cocktails and to each cell linecomponent in each cocktail (FIG. 76B).

The ability of the individual CRC vaccine component cell lines to induceIFNγ responses against themselves compared to the ability of thepotential CRC vaccine cocktails to induce IFNγ responses against theindividual cell lines was measured by IFNγ ELISpot as described inExamples 8 and 9. The data in FIG. 77 demonstrate that the cocktailsCRC-A, CRC-B, CRC-C, CRC-D, CRC-E, CRC-F, CRC-G and CRC-H (FIG. 76C) insome cases, trend toward or are significantly better stimulators ofantitumor immunity than the individual component cell lines and suggeststhat the breadth of response is increased by administering more than onecell line at a time.

The cells in the vaccine described herein were selected to express awide array of TAAs, including those known to be important specificallyfor CRC antitumor responses, such as CEA, and also TAAs known to beimportant for targets for CRC and other solid tumors, such as TERT. Asshown herein, to further enhance the array of TAAs, HuTu80 wastransduced with a gene encoding modPSMA and HCT-116 was also modified toexpress modTBXT, modWT1, and the 28 amino acids spanning the KRASmutations G12D and G12V respectively that result in an activatingmutated form of KRAS, as described herein. KRAS mutations occur inapproximately 35% to 45% of CRC patients. KRAS G12V and G12D are themost frequently occurring of multiple KRAS mutations in CRC patients.

PSMA was endogenously expressed in one of the six component cell linesat >1.0 FPKM as described below. TBXT and WT1 were not expressedendogenously in any of the six component cell lines at >1.0 FPKM (FIG.78A). The KRAS mutations G12D and G12V were not expressed endogenouslyby any of the six component cell lines. Endogenous expression of KRASmutations was determined using cBioPortal. The cell line data sets weresearched with the HGNC gene symbol (KRAS) and each cell line wassearched within the “mutations” data set. The KRAS G13D mutation, alsoexpressed frequently in CRC tumors, was endogenously expressed by HCT-15and HCT-116.

The mRNA expression of representative TAAs in the present vaccine areshown in FIG. 78A. The present vaccine has high expression of allidentified twenty commonly targeted and potentially clinically relevantTAAs for inducing a CRC antitumor response. Some of these TAAs are knownto be primarily enriched in CRC tumors and some can also induce animmune response to CRC and other solid tumors. RNA abundance of thetwenty prioritized CRC TAAs was determined in 365 CRC patient sampleswith expression data available for all TAAs as described above todetermine CSC marker expression patient samples. Fourteen of theprioritized CRC TAAs were expressed by 100% of samples, 15 TAAs wereexpressed by 94.5% of samples, 16 TAAs were expressed by 65.8% ofsamples, 17 TAAs were expressed by 42.2% of samples, 18 TAAs wereexpressed by 25.8% of samples, 19 TAAs were expressed by 11.5% ofsamples and 20 TAAs were expressed by 1.4% samples (FIG. 78B). The KRASG12D (n=40) or G12V (n=37) mutation were expressed by 21.1% (n=77) ofthe 365 CRC patient tumor samples. The KRAS G13D mutation, that isendogenously expressed by two component cell lines, was expressed by7.7% (n=28) of the 365 CRC patient tumor samples. Thus, provided hereinare two compositions comprising a therapeutically effective amount ofthree cancer cell lines, wherein the combination of the cell linesexpress at least 14 TAAs associated with a cancer of a subset of CRCcancer subjects intended to receive said composition.

Expression of the transduced antigens modPSMA (SEQ ID NO: 37; SEQ ID NO:38) by HuTu80 (FIG. 79A), and modTBXT (SEQ ID NO: 49; SEQ ID NO: 50)(FIG. 79B) and modWT1 (SEQ ID NO: 49; SEQ ID NO: 50) (FIG. 79C) byHCT-116 were detected by flow cytometry as described herein. The genesencoding KRAS G12D (SEQ ID NO: 49; SEQ ID NO: 50) (FIG. 89D) and G12V(SEQ ID NO: 49; SEQ ID NO: 50) (FIG. 79D) were detected by RT-PCR asdescribed in Example 29 herein. The genes encoding modTBXT, modWT1, KRASG12D and KRAS G12V are subcloned into the same lentiviral transfervector separated by furin cleavage sites SEQ ID: X). IFNγ production tothe transduced antigens is described herein.

Because of the need to maintain maximal heterogeneity of antigens andclonal subpopulations the comprise each cell line, the gene modifiedcell lines utilized in the present vaccine have been established usingantibiotic selection and flow cytometry and not through limitingdilution subcloning.

Based on the expression and immunogenicity data presented herein, thecell lines identified in Table 57 were selected to comprise the presentCRC vaccine.

TABLE 57 CRC vaccine cell lines and histology Cocktail Cell Line NameHistology A HCT-15 Colorectal Adenocarcinoma A HuTu-80 DuodenumAdenocarcinoma A LS411N Colorectal Adenocarcinoma B HCT-116 ColorectalCarcinoma B RKO Colorectal Carcinoma B DMS 53 Lung Small Cell Carcinoma

Reduction of CD276 Expression

The HCT-15, HuTu-80, LS411N, HCT-116, RKO and DMS 53 component celllines expressed CD276 and expression was knocked out by electroporationwith ZFN as described in Example 13 and elsewhere herein. Because it wasdesirable to maintain as much tumor heterogeneity as possible, theelectroporated and shRNA modified cells were not cloned by limitingdilution. Instead, the cells were subjected to multiple rounds of cellsorting by FACS as described in Example 13. Expression of CD276 wasdetermined as described in Example 29. Reduction of CD276 expression isdescribed in Table 58. These data show that gene editing of CD276 withZFN resulted in greater than 99.6% CD276-negative cells in all sixvaccine component cell lines.

TABLE 58 Reduction of CD276 expression Parental Cell Modified Cell lineLine MFI Cell Line MFI % Reduction CD276 HCT-15 6,737 26 99.6 HuTu-8010,389 0 100.0 LS411N 34,278 4 100.0 HCT-116 12,782 0 100.0 RKO 3,632 0100.0 DMS 53 11,928 24 99.8 MFI reported with isotype controlssubtracted

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12 werecompleted as described in Example 29.

shRNA Downregulates TGF-β Secretion

Following CD276 knockout, TGFβ1 and TGFβ2 secretion levels were reducedusing shRNA and resulting levels determined as described in Example 29.All parental cell lines in CRC vaccine-A secreted measurable levels ofTGFβ1 and HuTu80 also secreted a measurable level of TGFβ2. Of theparental cell lines in CRC vaccine-B, HCT-116 and RKO secretedmeasurable levels of TGFβ1. Reduction of TGFβ2 secretion by the DMS 53cell line is described in Example 5 and resulting levels determined asdescribed above.

The five component cell lines of CRC origin were transduced with TGFβ1shRNA to decrease secretion of TGFβ1 and increase expression of membranebound CD40L as described in Example 29. These cells are described by theclonal designation DK2. HuTu80 was subsequently transduced withlentiviral particles encoding TGFβ2 shRNA and GM-CSF (SEQ ID NO: 6)Example 29. These cells are described by the clonal designation DK6. Asdescribed in Example 26, DMS 53 was modified with shRNA to reducesecretion of TGFβ2 and not TGFβ1. These cells are described by theclonal designation DK4. The remaining cell lines were double modifiedwith TGFβ1 shRNA and TGFβ2 shRNA.

Table 59 shows the percent reduction in TGFβ1 and/or TGFβ2 secretion ingene modified component cell lines compared to unmodified, parental celllines. If TGFβ1 or TGFβ2 secretion was only detected in 1 of 16replicates run in the ELISA assay the value is reported without standarderror of the mean. Gene modification resulted in at least 49% reductionof TGFβ1 secretion. Gene modification of TGFβ2 resulted in at least 51%reduction in secretion of TGFβ2.

TABLE 59 TGF-β Secretion (pg/10⁶ cells/24 hr) in Component Cell LinesCell Line Cocktail Clone TGFβ1 TGFβ2 HCT-15 A Wild type 369 ± 69 21HCT-15 A DK2 189 ± 23 21 ± 5 HCT-15 A Percent reduction 49% NA HuTu-80 AWild type 2,529 ± 549  4,299 ± 821  HuTu-80 A DK6 327 ± 76 115 ± 42HuTu-80 A Percent reduction 87% 97% LS411N A Wild type  413 ± 125 * ≤9LS411N A DK2 89 ± 5  78 ± 13 LS411N A Percent reduction 78% NA HCT-116 BWild type 2,400 ± 250  * ≤8 HCT-116 B DK2 990 ± 72 * ≤8 HCT-116 BPercent reduction 59% NA RKO B Wild type  971 ± 120 * ≤6 RKO B DK2 206 ±10 * ≤11  RKO B Percent reduction 79% NA DMS 53 B Wild type 106 ± 10 486± 35 DMS 53 B DK4 219 ± 33 238 ± 40 DMS 53 B Percent reduction NA 51%DK6: TGFβ1/TGFβ2 double knockdown; DK4: TGFβ2 single knockdown; DK2:TGFβ1 single knockdown; * = estimated using LLD, not detected; NA = notapplicable

Based on a dose of 5×10⁵ of each component cell line, the total TGFβ1and TGFβ2 secretion by the modified CRC vaccine-A and CRC vaccine-B andrespective unmodified parental cell lines are shown in Table 60. Thesecretion of TGFβ1 by CRC vaccine-A was reduced by 82% and TGFβ2 by 95%pg/dose/24 hr. The secretion of TGFβ1 by CRC vaccine-B was reduced by59% and TGFβ2 by 49% pg/dose/24 hr.

TABLE 60 Total TGF-β Secretion (pg/dose/24 hr) in CRC vaccine-A and CRCvaccine-B Cocktail Clones TGFβ1 TGFβ2 A Wild type 1,656 2,165   DK2/DK6303 107 Percent reduction 82% 95% B Wild type 1,739 250 DK2/DK4 708 129Percent reduction 59% 49%

GM-CSF Secretion

The HuTu80 cell line was transduced with lentiviral particles containingboth TGFβ2 shRNA and the gene to express GM-CSF (SEQ ID NO: 6) under thecontrol of a different promoter. The HCT-15, LS411N, HCT-116 and RKOcell lines were transduced with lentiviral particles to only expressGM-CSF (SEQ ID NO: 7). DMS 53 was modified to secrete GM-CSF asdescribed in Example 24 and elsewhere herein. The results are shown inTable 61 and described below.

Secretion of GM-CSF increased at least 9,182-fold in all modifiedcomponent cell lines compared to unmodified, parental cell lines. In CRCvaccine-A component cell lines, secretion of GM-CSF increased29,500-fold by HCT-15 compared to the parental cell line (≤0.002 ng/10⁶cells/24 hr), 9,182-fold by HuTu80 compared to the parental cell line(≤0.011 ng/10⁶ cells/24 hr), and 36,250-fold by LS411N compared to theparental cell line (≤0.004 ng/10⁶ cells/24 hr). In CRC vaccine-Bcomponent cell lines secretion of GM-CSF increased 114,000-fold byHCT-116 compared to the parental cell line (≤0.003 ng/10⁶ cells/24 hr),43,667-fold by RKO compared to the parental cell line (≤0.003 ng/10⁶cells/24 hr) and 39,450-fold by DMS 53 compared to the parental cellline (≤0.004 ng/10⁶ cells/24 hr).

TABLE 61 GM-CSF Secretion in Component Cell Lines GM-CSF GM-CSF CellLine (ng/10⁶ cells/24 hr) (ng/dose/24 hr) HCT-15 59 ± 9 30 HuTu80 101 ±40 51 LS411N 145 ± 17 73 Cocktail A Total 305 154 HCT-116 342 ± 97 171RKO 131 ± 13 66 DMS 53 158 ± 15 79 Cocktail B Total 631 316

Based on a dose of 5×10⁵ of each component cell line, the total GM-CSFsecretion for CRC vaccine-A was 154 ng per dose per 24 hours. The totalGM-CSF secretion for CRC vaccine-B was 316 ng per dose per 24 hours. Thetotal GM-CSF secretion per dose was therefore 470 ng per 24 hours.

Membrane Bound CD40L (CD154) Expression

The component cell lines were transduced with lentiviral particles toexpress membrane bound CD40L as described above. The methods to detectexpression of CD40L by the five CRC cell line components are describedin Example 29. The methods used to modify DMS 53 to express CD40L aredescribed in Example 15. Evaluation of membrane bound CD40L by all sixvaccine component cell lines is described below. The results shown inFIG. 80 and described below demonstrate CD40L membrane expression wassubstantially increased in all six cell CRC vaccine component celllines.

FIG. 80 shows expression of membrane bound CD40L by the CRC vaccinecomponent cell lines. Membrane bound CD40L increased at least 669-foldin all component cell lines compared to unmodified, parental cell lines.In CRC vaccine-A component cell lines, expression of CD40L increased669-fold by HCT-15 (669 MFI) compared to the parental cell line (0 MFI),1,178-fold by HuTu80 (5,890 MFI) compared to the parental cell line (5MFI), and 4,703-fold by LS411N (4,703) compared to the parental cellline (0 MFI). In CRC vaccine-B component cell lines expression of CD40Lincreased 21,549-fold by HCT-116 compared to the parental cell line (0MFI), 7,107-fold by RKO compared to the parental cell line (0 MFI), and88,261-fold by DMS 53 compared to the parental cell line (0 MFI).

IL-12 Expression

The component cell lines were transduced with the IL-12 vector asdescribed in Example 17 and resulting IL-12 p70 expression determined asdescribed above and herein. The results are shown in Table 52 anddescribed below.

Secretion of IL-12 increased at least 10,200-fold in all component celllines modified to secrete IL-12 p70 compared to unmodified, parentalcell lines. In CRC vaccine-A component cell lines, secretion of IL-12increased 27,000-fold by HCT-15 compared to the parental cell line(≤0.001 ng/10⁶ cells/24 hr), 10,200-fold by HuTu80 compared to theparental cell line (≤0.005 ng/10⁶ cells/24 hr), and 13,000-fold byLS411N compared to the parental cell line (≤0.002 ng/10⁶ cells/24 hr).In CRC vaccine-B component cell lines expression of IL-12 increased186,000-fold by HCT-116 compared to the parental cell line (≤0.001ng/10⁶ cells/24 hr) and 43,000-fold by RKO compared to the parental cellline (≤0.001 ng/10⁶ cells/24 hr). DMS 53 was not modified to secreteIL-12.

TABLE 52 IL-12 secretion in component cell lines IL-12 IL-12 Cell Line(ng/10⁶ cells/24 hr) (ng/dose/24 hr) HCT-15 27 ± 3 14 HuTu80  51 ± 14 26LS411N 26 ± 6 13 Cocktail A Total 104 52 HCT-116 186 ± 16 93 RKO 43 ± 822 DMS 53 NA NA Cocktail B Total 229 115

Based on a dose of 5×10⁵ of each component cell line, the total IL-12secretion for CRC vaccine-A was 52 ng per dose per 24 hours. The totalIL-12 secretion for CRC vaccine-B was 115 ng per dose per 24 hours. Thetotal IL-12 secretion per dose was therefore 167 ng per 24 hours.

Stable Expression of modPSMA by the HuTu80 Cell Line

As described above, the cells in the vaccine described herein wereselected to express a wide array of TAAs, including those known to beimportant to CRC antitumor immunity. To further enhance the array ofantigens, the HuTu80 cell line that was modified to reduce the secretionof TGFβ1 and TGFβ2, reduce the expression of CD276, and to expressGM-CSF, membrane bound CD40L, and IL-12 was also transduced withlentiviral particles expressing the modPSMA antigen. The expression ofmodPSMA was characterized by flow cytometry. The cell line that wasmodified to reduce the secretion of TGFβ1 and TGFβ2, reduce theexpression of CD276, and to express GM-CSF, membrane bound CD40L, andIL-12 (antigen unmodified) and the cell line that was modified to reducethe secretion of TGFβ1 and TGFβ2, reduce the expression of CD276, and toexpress GM-CSF, membrane bound CD40L, IL-12 and modPSMA were stainedintracellularly with 0.06 μg/test anti-mouse IgG1 anti-PSMA antibody(AbCam ab268061, Clone FOLH1/3734) followed by 0.125 ug/testAF647-conjugated goat anti-mouse IgG1 antibody (Biolegend #405322). TheMFI of the isotype control stained PSMA unmodified and PSMA modifiedcells was subtracted from the MFI of cells stained PSMA. MFI wasnormalized to 100,000 events. Fold increase in antigen expression wascalculated as: (background subtracted modified MFI/background subtractedparental MFI). Expression of modPSMA increased in the modified cell line(756,908 MFI) 9.1-fold over that of the PSMA unmodified cell line(82,993 MFI) (FIG. 79A).

Stable Expression of modTBXT, modWT1, KRAS G12D and KRAS G12V by theHCT-116 Cell Line

As described above, the cells in the vaccine described herein wereselected to express a wide array of TAAs, including those known to beimportant to antitumor immunity. To further enhance the array ofantigens, the HCT-116 cell line that was modified to reduce thesecretion of TGFβ1, reduce the expression of CD276, and to expressGM-CSF, membrane bound CD40L and IL-12 was also transduced withlentiviral particles expressing the modTBXT, modWT1, KRAS G12V and KRASG12D antigens. The antigen unmodified and antigen modified cells werestained intracellular to detect the expression of each antigen asfollows. For the detection of modTBXT, cells were first stained withrabbit IgG1 anti-TBXT antibody (Abcam ab209665, Clone EPR18113) (0.06μg/test) followed by AF647-conjugated donkey anti-rabbit IgG1 antibody(Biolegend #406414) (0.125 μg/test). For the detection of modWT1, cellswere first stained with rabbit IgG1 anti-WT1 antibody (AbCam ab89901,Clone CAN-R9) (0.06 ug/test) followed by AF647-conjugated donkeyanti-rabbit IgG1 antibody (Biolegend #406414) (0.125 μg/test). The MFIof the isotype control stained cells was subtracted from the MFI of thecells stained for TBXT or WT1. MFI was normalized to 100,000 events.Fold increase in antigen expression was calculated as: (backgroundsubtracted modified MFI/background subtracted parental MFI). Expressionof modTBXT increased in the modified cell line (356,691 MFI)356,691-fold over that of the unmodified cell line (0 MFI) (FIG. 79B).Subtraction of the MFI of the isotype control from the MFI of the TBXTstained unmodified HCT-116 cell line resulted in negative value. Thefold increase of TBXT expression in the modified cell line wascalculated using 1 MFI. Expression of modWT1 by the modified cell line(362,698 MFI) increased 69.3-fold over the that of the unmodified cellline (5,235 MFI) (FIG. 79C).

Expression of KRAS G12D and KRAS G12V by HCT-116 was determined usingRT-PCR as described in Example 29 and herein. For KRAS G12D, the forwardprimer designed to anneal at the 2786-2807 base pair (bp) location inthe transgene (GAAGCCCTTCAGCTGTAGATGG (SEQ ID NO: 124)) and reverseprimer designed to anneal at 2966-2984 bp location in the transgene(CTGAATTGTCAGGGCGCTC (SEQ ID NO: 125)) and yield 199 bp product. ForKRAS G12V, the forward primer was designed to anneal at the 2861-2882 bplocation in the transgene (CATGCACCAGAGGAACATGACC (SEQ ID NO: 126)) andreverse primer designed to anneal at the 3071-3094 bp location in thetransgene (GAGTTGGATGGTCAGGGCAGAT (SEQ ID NO: 127)) and yield 238 bpproduct. Control primers for β-tubulin are described in Example 29. Geneproducts for both KRAS G12D and KRAS G12V were detected at the expectedsize, 199 bp and 238 bp, respectively (FIG. 79D). KRAS G12D mRNAincreased 3,127-fold and KRAS G12V mRNA increased 4,095-fold relative toparental controls (FIG. 79E).

Immune Responses to PSMA in CRC-Vaccine A

IFNγ responses to the PSMA were evaluated in the context of theCRC-vaccine A in four HLA diverse donors (n=4/donor) (Table 63 Donors 1,3, 5 and 6) as described in Example 29 and IFNγ responses determined byELISpot as described below. PSMA peptides, 15-mers overlapping by 9amino acids spanning the native antigen sequence, were purchased fromThermo Scientific Custom Peptide Service. PSMA specific IFNγ responseswere increased with the modified CRC vaccine-A (1,832±627 SFU) comparedto the parental, unmodified CRC vaccine-A (350±260 SFU) (n=4) (FIG.79F).

Immune Responses to TBXT, WT1, and KRAS Mutations in CRC-Vaccine B

IFNγ responses to TBXT, WT1, KRAS G12D and KRAS G12V antigens wereevaluated in the context of the CRC-vaccine B in four HLA diverse donors(n=4/donor) (Table 63. Donors 1, 3, 5 and 6) as described in Example 29.Peptides for were sourced as follows: TBXT (JPT, PM-BRAC), WT1 (JPT,PM-WT1), KRAS G12D and KRAS G12V 15-mers overlapping by 9 amino acids,were purchased from Thermo Scientific Custom Peptide Service. IFNγresponses to TBXT increased with the modified CRC vaccine-B (511±203SFU) compared to the unmodified CRC vaccine-B (154±111 SFU) (n=4) (FIG.79G). WT1 specific IFNγ responses significantly increased with themodified CRC vaccine-B (1,278±303 SFU) compared unmodified CRC vaccine-B(208±208 SFU) (p=0.027, Student's T test) (FIG. 79H). KRAS G12D specificIFNγ responses significantly increased with the modified CRC vaccine-B(1,716±420 SFU) compared unmodified CRC vaccine-B (153±153 SFU)(p=0.013, Student's T test) (FIG. 79I). KRAS G12V specific IFNγresponses significantly increased with the modified CRC vaccine-B(2,047±420 SFU) compared unmodified CRC vaccine-B (254±525 SFU)(p=0.018, Student's T test) (FIG. 79J).

TABLE 63 Healthy Donor MHC-I characteristics Donor # HLA-A HLA-B HLA-C 1*02:01*03:01 *08:01 *51:01 *07:01 *14:02 2 *30:02 *30:04 *15:10 *58:02*03:04 *06:02 3 *01:01 *30:01 *08:01 *13:02 *06:02 *07:01 4 *03:01*25:01 *17:02 *18:01 *07:02 *12:03 5 *02:05 *29:02 *15:01 *44:03 *03:04*16:01 6 *02:01*03:01 *18:01 *31:08 *07:01 *12:03

Cocktails Induce Immune Responses Against Relevant TAAs

The ability of the individual component cell lines and the two CRCvaccine cocktails to induce IFNγ production against relevant CRCantigens was measured by ELISpot as described in Example 29 using PBMCsfrom six HLA-diverse healthy donors (Table 63). Peptides for PSMA, WT1,TBXT, KRAS G12D and KRAS G12V were sourced as described above. Peptidesfor the remaining antigens were sourced as follows: Survivin(thinkpeptides, 7769_001-011), PRAME (Miltenyi Biotech, 130-097-286),STEAP (PM-STEAP1), TERT (JPT, PM-TERT), MUC1 (JPT, PM-MUC1), and CEACAM(CEA) (JPT, PM-CEA). Cells were then assayed for IFNγ secretion in theIFNγ ELISpot assay.

FIG. 81 demonstrates the CRC vaccine is capable of inducing antigenspecific IFNγ responses in six HLA-diverse donors that are significantlymore robust (30,480±9,980 SFU) compared to the unmodified parentalcontrols (6,470±3,361SFU) (p=0.009, Mann-Whitney U test) (n=8) (FIG.81A). CRC vaccine-A and CRC vaccine-B independently demonstrated antigenspecific responses significantly greater compared to parental controls.Specifically, CRC vaccine-A elicited 12,080±3,569 SFU compared to theunmodified controls (3,665±1,849 SFU) (p=0.041, Mann-Whitney U test)(FIG. 81B). For CRC vaccine-A, one donor responded to five antigens, onedonor responded to nine antigens, two donors responded to ten antigens,and two donors responded to eleven antigens. CRC vaccine-B (n=11antigens) elicited 15,417±4,127 SFU compared to parental controls(2,805±1,549 SFU) (p=0.004, Mann-Whitney U test) (FIG. 81C). For CRCvaccine-B (n=11 antigens), one donor responded to nine antigens, twodonors responded to ten antigens, and three donors responded to elevenantigens. The CRC vaccine (vaccine-A and vaccine-B) induced IFNγproduction to ten antigens in two of six donors and all eleven antigensin four of six donors (FIG. 82) (Table 64). Thus, provided herein aretwo compositions comprising a therapeutically effective amount of threecancer cell lines (e.g., a unit dose of six cell lines) wherein saidunit dose is capable of eliciting an immune response 4.7-fold greaterthan the unmodified composition specific to at least ten TAAs expressedin CRC patient tumors. CRC vaccine A increased IFNγ responses to atleast five TAAs 4.1-fold and CRC vaccine-B increased IFNγ responses toat least nine TAAs 5.5-fold.

IFNγ responses to TAAs induced by CRC vaccine-A and CRC vaccine-B weremore robust than compared to responses induced by the individualmodified CRC cell line components. Specifically, CRC vaccine-Aassociated responses against the eleven assayed antigens (18,910±8,852SFU) were greater than responses induced by modified HCT-15(11,255±6,354 SFU), HuTu80 (7,332±2,814 SFU) and LS411N (8,277±3,187SFU). Similarly, CRC vaccine-B associated responses against the elevenassayed antigens (17,635±6,056 SFU) were greater than responses inducedby modified HCT-116 (11,984±5,085 SFU) and RKO (10,740±5,216 SFU) (FIG.83).

TABLE 64 IFNγ Responses to TAAs induced by the unmodified and modifiedCRC vaccine Donor Unmodified (SFU ± SEM) Modified (SFU ± SEM) (n = 4)CRC vaccine-A CRC vaccine-B CRC vaccine CRC vaccine-A CRC vaccine-B CRCvaccine 1 6,101 ± 2,763 2,659 ± 1,128 8,760 ± 3,640 3,969 ± 2,029 11,498± 3,813  15,466 ± 5,590  2 3,694 ± 1,363 3,699 ± 1,868 7,394 ± 3,2175,465 ± 2,522 8,543 ± 4,763 14,008 ± 7,258  3 11,488 ± 1,912  9,910 ±3,165 21,398 ± 4,907  43,448 ± 7,892  35,693 ± 4,638  79,140 ± 11,908 4100 ± 50  388 ± 130 488 ± 84  9,276 ± 3,150 13,419 ± 5,196  22,694 ±7,650  5 0 ± 0 0 ± 0 0 ± 0 12,666 ± 5,766  10,052 ± 6,559  22,718 ±11,181 6 608 ± 334 173 ± 103 781 ± 436 15,557 ± 3,291  13,296 ± 2,843 28,853 ± 5,346 

Based on the disclosure and data provided herein, a whole cell vaccinefor Colorectal Carcinoma comprising the six cancer cell lines, sourcedfrom ATCC, HCT-15 (ATCC, CCL-225), HuTu80 (ATCC, HTB-40), LS411N (ATCC,CRL-2159), HCT-116 (ATCC, CCL-247), RKO (ATCC, CRL-2577) and DMS 53(ATCC, CRL-2062) is shown in Table 65. The cell lines represent fivecolorectal cell lines and one small cell lung cancer (SCLC) cell line(DMS 53 ATCC CRL-2062). The cell lines have been divided into twogroupings: vaccine-A and vaccine-B. Vaccine-A is designed to beadministered intradermally in the upper arm and vaccine-B is designed tobe administered intradermally in the thigh. Vaccine A and B togethercomprise a unit dose of cancer vaccine.

TABLE 65 Cell line nomenclature and modifications TGFβ1 TGFβ2 CocktailCell Line KD KD CD276 KO GM-CSF CD40L IL-12 TAA(s) A HCT-15 X ND X X X XND A HuTu80 X X X X X X X A LS411N X ND X X X X ND B HCT-116 X ND X X XX X B RKO X ND X X X X ND B DMS 53* ND X X X X ND ND ND = Not done.*Cell lines identified as CSC-like cells.

Where indicated in the above table, the genes for the immunosuppressivefactors transforming growth factor-beta 1 (TGFβ1) and transforminggrowth factor-beta 2 (TGFβ2) have been knocked down using shRNAtransduction with a lentiviral vector. The gene for CD276 has beenknocked out by electroporation using zinc-finger nuclease (ZFN). Thegenes for granulocyte macrophage—colony stimulating factor (GM-CSF),IL-12, CD40L, modPSMA (HuTu80), modTBXT (HCT-116), modWT1 (HCT-116),KRAS G12D (HCT-116) and KRAS G12V (HCT-116) have been added bylentiviral vector transduction.

Provided herein are two compositions comprising a therapeuticallyeffective amount of three cancer cell lines, a unit dose of six cancercell lines, modified to reduce the expression of at least twoimmunosuppressive factors and to express at least two immunostimulatoryfactors. One composition, CRC vaccine-A, was modified to increase theexpression of one TAA, modPSMA, and the second composition, CRCvaccine-B, was modified to expresses four TAAs, modTBXT, modWT1, KRASG12D and KRAS G12V. The unit dose of six cancer cell lines expresses atleast fifteen TAAs in CRC patient tumors and induces IFNγ responses4.7-fold greater than the unmodified composition components.

Example 31: Preparation of Prostate Cancer (PCa) Vaccine

This Example demonstrates that reduction of TGFβ1, TGFβ2, and CD276expression with concurrent overexpression of GM-CSF, CD40L, and IL-12 ina vaccine composition of two cocktails, each cocktail composed of threecell lines for a total of 6 cell lines, significantly increased themagnitude of cellular immune responses to at least 10 PCa-associatedantigens in an HLA-diverse population. As described herein, the firstcocktail, PCa vaccine-A, is composed of cell line PC3 that was alsomodified to express modTBXT and modMAGEC2, cell line NEC8, and cell lineNTERA-2c1-D1. The second cocktail, PCa vaccine-B, is composed of cellline DU145 that was also modified to express modPSMA, cell line LNCaP,and cell line DMS 53. The six component cell lines collectively expressat least twenty-two antigens that can provide an anti-PCa tumorresponse.

Identification of PCa Vaccine Components

Initial cell line selection criteria identified sixteen vaccinecomponent cell lines for potential inclusion in the PCa vaccine.Additional selection criteria were applied to narrow the fourteencandidate cell lines to six cell lines for further evaluation inimmunogenicity assays. These criteria included: endogenous PCaassociated antigen expression, lack of expression of additionalimmunosuppressive factors, such as IL-10 or IDO1, ethnicity and age ofthe patient from which the cell line was derived, if the cell line wasderived from a primary tumor or metastatic site, and histologicalsubtype.

Expression of TAAs by candidate component cell lines was determined byRNA expression data sourced from the Broad Institute Cancer Cell LineEncyclopedia (CCLE) and from the European Molecular BiologyLaboratory-European Bioinformatics Institute (EMBL-EBI) for NCCIT, NEC8and NTERA-2c1-D1. The HGNC gene symbol was included in the CCLE searchand mRNA expression was downloaded for each TAA. Expression of a TAA bya cell line was considered positive if the RNA-seq value was greaterthan one (CCLE, FPKM) or zero (EMBL-EBI, TPM). Six of the fourteen PCavaccine candidate components were identified for further evaluation:PC3, DU145, LNCaP, NCCIT, NEC8 and NTERA-2c1-D1 based on the selectioncriteria described above. The six candidate component cell linesexpressed twelve to nineteen TAAs (FIG. 84). As described herein, theCSC-like cell line DMS 53 is included as one of the six cell lines andexpressed sixteen PCa TAAs.

Immunogenicity of the unmodified PCa individual component cell linecandidates was evaluated by IFNγ ELISpot as described in Example 9 forfour HLA diverse healthy donors (n=4 per donor). HLA-A and HLA-B allelesfor the donors were as follows: Donor 1, A*02:01 B*35:01 and A*31:01B*35:03; Donor 2, A*02:02 B*15:03 and A*30:02 B*57:03; Donor 3, A*02:01B*40:01 and A*30:01 B*57:01; Donor 4, A*24:02 B*18:01 and A*02:01B*15:07. PC3 (3,409±672 SFU) and DU145 (1,497±231 SFU) were moreimmunogenic than LNCaP (428±204 SFU), NCCIT (25±11 SFU), NEC8 (80±47SFU) and NTERA-2c1-D1 (188±93 SFU) (FIG. 86A). NCCIT was poorlyimmunogenic and excluded from further analysis. PC3 and DU145 wereselected to be included in vaccine cocktail A and vaccine cocktail B,respectively, as described further herein.

Immunogenicity of five selected PCa cell lines and the CSC cell line DMS53 was evaluated in two different combinations of three component celllines (FIG. 86C). IFNγ responses were determined against the threecomponent cell lines within the two potential vaccine cocktails by IFNγELISpot as described in Example 8 in five HLA diverse healthy donors(n=4 per donor). HLA-A and HLA-B alleles for the donors were as follows:Donor 1, A*02:01 B*08:01 and A*03:01 B*51:01; Donor 2, A*30:02 B*18:01and A*30:04 B*58:02, Donor 3, A*02:01 B*18:01 and A*25:01 B*27:05; Donor4, A*03:01 B*07:02 and A*25:01 B*18:01; Donor 5, A*02:01 B*07:02 andA*33:01 B*14:02. IFNγ responses were detected for both cocktails and toeach cell line component in each cocktail. (FIG. 86B).

The ability of the individual PCa vaccine component cell lines to induceIFNγ responses against themselves compared to the ability of thepotential PCa vaccine cocktails to induce IFNγ responses against theindividual cell lines was also measured by IFNγ ELISpot as described inExamples 8 and 9. IFNγ responses to the NEC8 cell line in PCa-A(1,963±863 SFU) were significantly increased compared to responses thecell line alone (283±101 SFU) (Mann-Whitney U test, p=0.032). Similarly,IFNγ responses to the NTERA-2c1-D1 cell line in PCa-A (630±280 SFU) weresignificantly increased compared to responses the cell line alone(283±101 SFU) (Mann-Whitney U test, p=0.032). IFNγ responses to theLNCaP cell line in PCa-B (624±254 SFU) were significantly increasedcompared to responses the cell line alone (139±111 SFU) (Mann-Whitney Utest, p=0.032). The data in FIG. 86D demonstrate that the cocktailsPCa-A and PCa-B, in some cases, trend toward or are significantly betterstimulators of antitumor immunity than the individual component celllines and suggest that the breadth and magnitude of response isincreased by administering multiple cell lines with different HLAsupertypes. Specifically, PCa-A cell lines are the following HLAsupertypes: PC3, A01 A24 and B07; NTERA-2c1-D1, A01, B08, and B44. TheHLA type of NEC8 is unavailable. PCa-B cell lines are the following HLAsupertypes: DU145, A03, B44, and B58; LNCaP, A01, A02 B08, B44; DMS 53,A03, B08 and B07. The data above supports that HLA mismatch of celllines comprising cocktails can improve immune responses to individualcell line components.

The cells in the vaccine described herein were selected to express awide array of TAAs, including those known to be important specificallyfor PCa antitumor responses, such as PSA or PAP, and also TAAs known tobe important for targets for PCa and other solid tumors, such TERT. Asshown herein, to further enhance the array of TAAs, DU145 was transducedwith a gene encoding modPSMA and PC3 was modified to express modTBXT andmodMAGEC2. PSMA was endogenously expressed in three of the six componentcell lines at >1.0 FPKM or >0 TPM. TBXT and MAGEC2 were endogenously intwo of the six component cell lines at >1.0 FPKM or >0 TPM (FIG. 84).

Expression of the transduced antigens modTBXT (FIG. 87A) and modMAGEC2(FIG. 87B) (SEQ ID NO: 45; SEQ ID NO: 46) by PC3, and modPSMA (SEQ IDNO: 37; SEQ ID NO: 38) by DU145 (FIG. 87C) were detected by flowcytometry or RT-PCR described in Example 29 and herein. The genesencoding modTBXT and modMAGEC2 are encoded in the same lentiviraltransfer vector separated by a furin cleavage site.

Because of the need to maintain maximal heterogeneity of antigens andclonal subpopulations the comprise each cell line, the gene modifiedcell lines utilized in the present vaccine have been established usingantibiotic selection and flow cytometry and not through limitingdilution subcloning.

The mRNA expression of twenty-two representative TAAs in the presentvaccine are shown in FIG. 84. NCCIT is the only cell line in FIG. 84that is not included in the present vaccine. The present vaccine hashigh expression of all identified twenty-two commonly targeted andpotentially clinically relevant TAAs for inducing a PCa antitumorresponse. Some of these TAAs are known to be primarily enriched in PCatumors and some can also induce an immune response to PCa and othersolid tumors. RNA abundance of the twenty-two prioritized PCa TAAs wasdetermined in 460 PCa patient samples (FIG. 85A) with expression dataavailable for all TAAs as described in Example 29. Eighteen of theprioritized PCa TAAs were expressed by 100% of samples, 19 TAAs wereexpressed by 99.3% of samples, 20 TAAs were expressed by 75.4% ofsamples, 21 TAAs were expressed by 21.1% of samples, 22 TAAs wereexpressed by 1.5% of samples (FIG. 85B). Provided herein are twocompositions comprising a therapeutically effective amount of threecancer cell lines, wherein the combination of the cell lines comprisescells express at least 18 TAAs associated with a cancer of a subset ofPCa cancer subjects intended to receive said composition. Based on theexpression and immunogenicity data presented herein, the cell linesidentified in Table 66 were selected to comprise the present PCavaccine.

TABLE 66 PCa vaccine cell lines and histology Cell Line Cocktail NameHistology A PC3 Prostate Carcinoma derived from metastatic site (bone) ANEC8 Testicular Germ Cell Tumor A NTERA-2cl-D1 Testis EmbryonalCarcinoma derived from metastatic site (lung) B DU145 Prostate Carcinomaderived from metastatic site (bone) B LNCaP Prostate Carcinoma derivedfrom metastatic site (lymph node) B DMS 53 Lung Small Cell Carcinoma

Reduction of CD276 Expression

The PC3, NEC8, NTERA-2c1-D1, DU145, LNCaP and DMS 53 component celllines expressed CD276 and expression was knocked out by electroporationwith ZFN as described in Example 13 and elsewhere herein. Because it wasdesirable to maintain as much tumor heterogeneity as possible, theelectroporated and shRNA modified cells were not cloned by limitingdilution. Instead, the cells were subjected to multiple rounds of cellsorting by FACS as described in Example 13. Expression of CD276 wasdetermined as described in Example 29. Reduction of CD276 expression isdescribed in Table 67. These data show that gene editing of CD276 withZFN resulted in greater than 98.7% CD276-negative cells in all sixvaccine component cell lines.

TABLE 67 Reduction of CD276 expression Parental Modified Cell line CellLine MFI Cell Line MFI % Reduction CD276 PC3 6,645 0 100.0 NEC8 6,317 3399.5 NTERA-2cl-D1 7,240 95 98.7 DU145 8,461 8 99.9 LNCaP 41,563 3 99.9DMS 53 11,928 24 99.8 MFI reported with isotype controls subtracted

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12 werecompleted as described in Example 29.

shRNA Downregulates TGF-β Secretion

Following CD276 knockout, TGFβ1 and TGFβ2 secretion levels were reducedusing shRNA and resulting levels determined as described in Example 29.The PC3 and NEC8 parental cell lines in PCa vaccine-A secretedmeasurable levels of TGFβ1. PC3 also secreted a measurable level ofTGFβ2. NEC8 secreted relatively low levels of TGFβ1 and did not secretemeasurable levels of TGFβ2. NTERA-2c1-D1 did not secreted measurablelevels of TGFβ1 or TGFβ2. Of the parental cell lines in PCa vaccine-B,DU145 secreted measurable, but relatively low levels of TGFβ1 and TGFβ2,and LNCaP did not secrete measurable levels of TGFβ1 or TGFβ2. Reductionof TGFβ2 secretion by the DMS 53 cell line is described in Example 26and resulting levels determined as described above.

The PC3 component cell line was transduced with TGFβ1 shRNA to decreasesecretion of TGFβ1 and increase expression of membrane bound CD40L asdescribed in Example 29 and was subsequently transduced with lentiviralparticles encoding TGFβ2 shRNA and GM-CSF (SEQ ID NO: 6) Example 29.These cells are described by the clonal designation DK6. As described inExample 26, DMS 53 was modified with shRNA to reduce secretion of TGFβ2and not TGFβ1. These cells are described by the clonal designation DK4.The remaining cell lines were not modified with TGFβ1 shRNA or TGFβ2shRNA.

Table 68 shows the percent reduction in TGFβ1 and/or TGFβ2 secretion ingene modified component cell lines compared to unmodified, parental,cell lines. If TGFβ1 or TGFβ2 secretion was only detected in 1 of 16replicates run in the ELISA assay the value is reported without standarderror of the mean. Gene modification resulted in 82% reduction of TGFβ1secretion. Gene modification of TGFβ2 resulted in at least 51% reductionin secretion of TGFβ2.

TABLE 68 TGF-β Secretion (pg/10⁶ cells/24 hr) in Component Cell LinesCell Line Cocktail Clone TGFβ1 TGFβ2 PC3 A Wild type 686 ± 93 3,878 ±556  PC3 A DK6  122 ± 119 382 ± 89 PC3 A Percent reduction 82% 90% NEC8A Wild type  97 ± 26  * ≤4 NEC8 A NA NA NA NEC8 A Percent reduction NANA NTERA-2cl-D1 A Wild type * ≤304  * ≤138  NTERA-2cl-D1 A NA NA NANTERA-2cl-D1 A Percent reduction NA NA DU145 B Wild type 161 ± 28 435 ±64 DU145 B NA NA NA DU145 B Percent reduction NA NA LNCaP B Wild type *≤63 * ≤28 LNCaP B NA NA NA LNCaP B Percent reduction NA NA DMS 53 B Wildtype 106 ± 10 486 ± 35 DMS 53 B DK4 NA 238 ± 40 DMS 53 B Percentreduction NA 51% DK6: TGFβ1/TGFβ2 double knockdown; DK4: TGFβ2 singleknockdown; DK2: TGFβ1 single knockdown; * = estimated using LLD, notdetected; NA = not applicable

Based on a dose of 5×10⁵ of each component cell line, the total TGFβ1and TGFβ2 secretion by the modified PCa vaccine-A and PCa vaccine-B andrespective unmodified parental cell lines are shown in Table 69. Thesecretion of TGFβ1 by PCa vaccine-A was reduced by 52% pg/dose/24 hr andTGFβ2 by 87% pg/dose/24 hr. The secretion of TGFβ2 by PCa vaccine-B wasreduced by 26% pg/dose/24 hr.

TABLE 69 Total TGF-β Secretion (pg/dose/24 hr) in PCa vaccine-A and PCavaccine-B Cocktail Clones TGFβ1 TGFβ2 A Wild type 544 2,010   DK6 262262 Percent reduction 52% 87% B Wild type 166 475 DK4 NA 351 Percentreduction NA 26%

GM-CSF Secretion

The PC3 cell line was transduced with lentiviral particles containingboth TGFβ2 shRNA and the gene to express GM-CSF (SEQ ID NO: 6) under thecontrol of a different promoter. The NEC8, NTERA-2c1-D1, DU145 and LNCaPcell lines were transduced with lentiviral particles to only expressGM-CSF (SEQ ID NO: 7). DMS 53 was modified to secrete GM-CSF asdescribed in Example 24 and elsewhere herein. The results are shown inTable 70 and described below.

Secretion of GM-CSF increased at least 68-fold in all modified componentcell lines compared to unmodified, parental cell lines. In PCa vaccine-Acomponent cell lines, secretion of GM-CSF increased 67,987-fold by PC3compared to the parental cell line (≤0.003 ng/10⁶ cells/24 hr),128,543-fold by NEC-8 compared to the parental cell line (≤0.002 ng/10⁶cells/24 hr), and 68-fold by NTERA-2c1-D1 compared to the parental cellline (≤0.059 ng/10⁶ cells/24 hr). In PCa vaccine-B component cell linessecretion of GM-CSF increased 119,645-fold by DU145 compared to theparental cell line (≤0.003 ng/10⁶ cells/24 hr), 10,151-fold by LNCaPcompared to the parental cell line (≤0.012 ng/10⁶ cells/24 hr) and39,450-fold by DMS 53 compared to the parental cell line (≤004 ng/10⁶cells/24 hr).

TABLE 70 GM-CSF Secretion in Component Cell Lines GM-CSF GM-CSF CellLine (ng/10⁶ cells/24 hr) (ng/dose/24 hr) PC3 187 ± 16 94 NEC-8 208 ± 9 104 NTERA-2cl-D1   4 ± 0.2 2 Cocktail A Total 399 200 DU145 386 ± 71 193LNCaP 124 ± 11 62 DMS 53 158 ± 15 79 Cocktail B Total 668 334

Based on a dose of 5×10⁵ of each component cell line, the total GM-CSFsecretion for PCa vaccine-A was 200 ng per dose per 24 hours. The totalGM-CSF secretion for PCa vaccine-B was 334 ng per dose per 24 hours. Thetotal GM-CSF secretion per dose was therefore 534 ng per 24 hours.

Membrane Bound CD40L (CD154) Expression

The component cell lines were transduced with lentiviral particles toexpress membrane bound CD40L vector as described above. The methods todetect expression of CD40L by the five PCa cell line components aredescribed in Example 29. The methods used to modify DMS 53 to expressCD40L are described in Example 15. Evaluation of membrane bound CD40L byall six vaccine component cell lines is described below. The resultsshown in FIG. 88 and described below demonstrate CD40L membraneexpression was substantially increased in all six cell PCa vaccinecomponent cell lines.

Expression of membrane bound CD40L by the PCa vaccine cell lines isshown in FIG. 88. Membrane-bound CD40L expression increased at least9,019-fold in all component cell lines compared to unmodified, parentalcell lines. In PCa vaccine-A component cell lines, expression of CD40Lincreased 9,019-fold by PC3 (9,019 MFI) compared to the parental cellline (0 MFI), 11,571-fold by NEC8 (11,571 MFI) compared to the parentalcell line (0 MFI), and 15,609-fold by NTERA-2c1-D1 (15,609 MFI) comparedto the parental cell line (0 MFI). In PCa vaccine-B component cell linesexpression of CD40L increased 18,699-fold by DU145 compared to theparental cell line (0 MFI), 30,243-fold by LNCaP compared to theparental cell line (0 MFI), and 88,261-fold by DMS 53 compared to theparental cell line (0 MFI).

IL-12 Expression

The component cell lines were transduced with the IL-12 vector asdescribed in Example 17 and resulting IL-12 p70 expression determined asdescribed above and herein. The results are shown in Table 71 anddescribed below.

Secretion of IL-12 increased at least 507-fold in all component celllines modified to secrete IL-12 p70 compared to unmodified, parentalcell lines. In PCa vaccine-A component cell lines, secretion of IL-12increased 42,727-fold by PC3 compared to the parental cell line (≤0.001ng/10⁶ cells/24 hr), 30,769-fold by NEC8 compared to the parental cellline (≤0.001 ng/10⁶ cells/24 hr), and 507-fold by NTERA-2c1-D1 comparedto the parental cell line (≤0.024 ng/10⁶ cells/24 hr). In PCa vaccine-Bcomponent cell lines expression of IL-12 increased 13,178-fold by DU145compared to the parental cell line (≤0.001 ng/10⁶ cells/24 hr) and3,901-fold by LNCaP compared to the parental cell line (≤0.005 ng/10⁶cells/24 hr). DMS 53 was not modified to secrete IL-12.

TABLE 71 IL-12 secretion in component cell lines IL-12 IL-12 Cell Line(ng/10⁶ cells/24 hr) (ng/dose/24 hr) PC3  47 ± 24 24 NEC-8 20 ± 3 10NTERA-2cl-D1 12 6 Cocktail A Total 79 40 DU145 17 ± 4 9 LNCaP 19 ± 6 10DMS 53 NA NA Cocktail B Total 36 19

Based on a dose of 5×10⁵ of each component cell line, the total IL-12secretion for PCa vaccine-A was 40 ng per dose per 24 hours. The totalIL-12 secretion for PCa vaccine-B was 19 ng per dose per 24 hours. Thetotal IL-12 secretion per dose was therefore 59 ng per 24 hours.

Stable Expression of modTBXT and modMAGEC2 by the PC3 Cell Line

As described above, the cells in the vaccine described herein wereselected to express a wide array of TAAs, including those known to beimportant to antitumor immunity. To further enhance the array ofantigens, the PC3 cell line that was modified to reduce the secretion ofTGFβ1 and TGFβ2, reduce the expression of CD276, and to express GM-CSF,membrane bound CD40L and IL-12 was also transduced with lentiviralparticles expressing the modTBXT and modMAGEC2 antigens. The genesencoding the modTBXT and modMAGEC2 antigens are linked by a furincleavage site (SEQ ID NO: 45, SEQ ID NO: 46).

The expression of modTBXT by PC3 was characterized by flow cytometry.For the detection of modTBXT expression cells were first stainedintracellular with rabbit IgG1 anti-TBXT antibody (Abcam ab209665, CloneEPR18113) (0.06 μg/test) followed by AF647-conjugated donkey anti-rabbitIgG1 antibody (Biolegend #4406414) (0.125 μg/test). Expression ofmodTBXT increased in the modified cell line (1,209,613 MFI)1,209,613-fold over that of the unmodified cell line (0 MFI) (FIG. 87A).The expression of modMAGEC2 by PC3 was determined using RT-PCR asdescribed in Example 29 and herein. The forward primer designed toanneal at the 604-631 base pair (bp) location in the transgene(GATCACTTCTGCGTGTTCGCTAACACAG (SEQ ID NO: 128)) and reverse primerdesigned to anneal at the 1072-1094 bp location in the transgene(CTCATCACGCTCAGGCTCTCGCT (SEQ ID NO: 129)) and yield 491 bp product.Control primers and resulting product for β-tubulin are described inExample 29. The gene product for MAGEC2 was detected at the expectedsize (FIG. 97B). modMAGEC2 mRNA increased 3,914-fold relative to theparental control (FIG. 87B).

Stable Expression of modPSMA by the DU145 Cell Line

The DU145 cell line that was modified to reduce the expression of CD276,and to express GM-CSF, membrane bound CD40L, and IL-12 was alsotransduced with lentiviral particles expressing the modPSMA antigen (SEQID NO: 37, SEQ ID NO: 38). The expression of modPSMA was characterizedby flow cytometry. Antigen unmodified and antigen modified cells werestained intracellular with 0.06 μg/test anti-mouse IgG1 anti-PSMAantibody (AbCam ab268061, Clone FOLH1/3734) followed by 0.125 ug/testAF647-conjugated goat anti-mouse IgG1 antibody (Biolegend #405322).Expression of modPSMA increased in the modified cell line (249,632 MFI)6-fold over that of the parental cell line (42,196 MFI) (FIG. 87C).

Immune Responses to TBXT and MAGEC2 in PCa Vaccine-A

IFNγ responses to TBXT and MAGEC2 antigens were evaluated in the contextof the modified PCa vaccine-A as described in Example 29, and herein, inseven HLA diverse donors (n=4/donor. The HLA-A, HLA-B, and HLA-C allelesfor each of the seven donors are shown in Table 72. IFNγ responses toTBXT were determined by ELISpot using 15-mers peptides overlapping by 11amino acids (JPT, PM-BRAC) spanning the entire length of the native TBXTantigen. IFNγ responses to TBXT significantly increased with themodified PCa vaccine-B (605±615 SFU) compared to the unmodified PCavaccine-A (73±70 SFU) (p=0.033, Mann-Whitney U test) (n=7) (FIG. 87D).IFNγ responses to MAGEC2 were determined by ELISpot using 15-merspeptides overlapping by 9 amino acids spanning the entire length of thenative antigen, purchased from Thermo Scientific Custom Peptide Service.IFNγ responses to MAGEC2 significantly increased with the modified PCavaccine-B (697±536 SFU) compared to the unmodified PCa vaccine-B (SFU)(p=0.018, Mann-Whitney U test) (n=7) (FIG. 87E).

Immune Responses to PSMA in PCa-Vaccine B

IFNγ responses to the PSMA antigen were evaluated in the context of thePCa-vaccine B as described in Example 29, and herein, in seven HLAdiverse donors (n=4/donor) (Table 72). IFNγ responses determined byELISpot as described in Example 29. PSMA peptides, 15-mers overlappingby 9 amino acids spanning the native antigen sequence, were purchasedfrom Thermo Scientific Custom Peptide Service. PSMA specific IFNγresponses with the were significantly increased with the modified PCavaccine-B (1,580±847 SFU) compared to the parental, unmodified PCavaccine-A (327±33 SFU) (p=0.011, Mann-Whitney U test) (n=7) (FIG. 87F).

TABLE 72 Healthy Donor MHC-I characteristics Donor # HLA-A HLA-B HLA-C 1*02:01 *03:01 *08:01 *51:01 *07:01 *14:02 2 *30:02 *30:01 *15:10 *58:02*03:04 *06:02 3 *03:01 *32:01 *07:02 *15:17 *07:01 *07:02 4 *03:01*25:01 *07:02 *18:01 *07:02 *12:03 5 *02:01 *33:01 *07:02 *14:02 *07:02*08:02 6 *01:01 *30:01 *08:01 *13:02 *06:02 *07:01 7 *26:01 *68:02*08:01 *15:03 *03:04 *12:03

Cocktails Induce Immune Responses Against Relevant TAAs

The ability of the two PCa vaccine cocktails to induce IFNγ productionagainst relevant PCa antigens was measured by ELISpot. PBMCs from sevenHLA-diverse healthy donors (Table 72) were co-cultured with the PCAvaccine-A or PCa vaccine-B cocktails for 6 days prior to stimulationwith autologous DCs loaded with TAA-specific specific peptide poolscontaining known MHC-I restricted epitopes. Peptides for stimulation ofCD14-PBMCs for detection of IFNγ responses to TBXT, MAGEC2 and PSMA aredescribed above. Additional 15-mer overlapping by 11 amino acid peptidepools were sourced as follows: TERT (JPT, PM-TERT), Survivin(thinkpeptides, 7769_001-011), HER2 (JPT, PM-ERB_ECD), STEAP(PM-STEAP1), MUC1 (JPT, PM-MUC1), PAP (JPT, PM-PAP), and PSA (JPT,PM-PSA). Cells were then assayed for IFNγ secretion in the IFNγ ELISpotassay.

FIG. 89 demonstrates the PCa vaccine is capable of inducing antigenspecific IFNγ responses in seven HLA-diverse donors to ten PCa antigensthat are significantly more robust (19,982±5,480 SFU) compared to theunmodified parental controls (3,259±1,046 SFU) (p=0.011, Mann-Whitney Utest) (n=7) (FIG. 89A). The unit dose of PCa vaccine-A and PCa vaccine-Belicited IFNγ responses to eight antigens in one of seven donors and tenantigens in six of the seven donors. PCa vaccine-A and PCa vaccine-Bindependently demonstrated antigen specific responses significantlygreater compared to parental controls. For PCa vaccine-A, one donorresponded to three antigens, one donor responded to eight antigens, onedonor responded to nine antigens, and four donors responded tenantigens. Specifically, PCa vaccine-A elicited 9,412±6,170 SFU comparedto the unmodified controls (1,430±911 SFU) (p=0.026, Mann-Whitney Utest) (FIG. 89B). For PCa vaccine-B, one donor responded to sixantigens, three donors responded to nine antigens, and three donorsresponded to ten antigens. PCa vaccine-B elicited 10,570±2,913 SFUcompared to parental controls (1,830±371 SFU) (p=0.004, Mann-Whitney Utest) (FIG. 89C). The PCA vaccine (vaccine-A and vaccine-B) induced IFNγproduction to nine antigens in one of seven donors and all ten antigensin six of seven donors (FIG. 90) (Table 73). Described above are twocompositions comprising a therapeutically effective amount of threecancer cell lines, a unit dose of six cell lines, wherein said unit doseis capable of eliciting an immune response 6.1-fold greater than theunmodified composition specific to at least eight TAAs expressed in PCApatient tumors. PCA vaccine-A increased IFNγ responses to at least threeTAAs 6.6-fold and PCA vaccine-B increased IFNγ responses 5.8-fold to atleast six TAAs.

The ability of the individual modified PCa vaccine component cell linesto induce IFNγ responses against matched unmodified cell line componentswas measured by IFNγ ELISpot as described in Examples 8 and 9 for fourHLA diverse donors (n=4/donor) (Table 73. Donors 1, 2, 4 and 5). IFNγresponses were detected against parental unmodified cell lines for bothcocktails and each modified cell line component in each cocktail. Therewas a trend towards increased IFNγ production for PCa vaccine-A and PCavaccine-B compared to individual modified cell lines, but this trend didnot reach statistical significance likely due to the low n of Donors(n=4) Mann Whitney U test for all comparisons) (FIG. 91A).

There was a significant difference in IFNγ production between PCavaccine-A and the individual modified cell line components (p=0.036,Kruskal Wallis test). Specifically, PCa vaccine-A induced significantlygreater IFNγ production (5,685±2,060 SFU) than the modified NTERA-2c1-D1(253±136) (p=0.019) component cell line but not the NEC8 (1,151±735 SFU)(p=0.307) and PC3 component cell line (1,898±947 SFU) (p=0.621)(post-hoc Dunn's test for multiple comparisons) (FIG. 91B). There wasalso a significant difference in IFNγ production between PCa vaccine-Band the individual modified cell line components (p=0.006, KruskalWallis test). Specifically, PCa vaccine-B induced significantly greaterIFNγ production (5,686±1,866 SFU) than the modified LNCaP (240±122 SFU)(p=0.043) and DMS 53 (222±113) (p=0.028) component cell lines but notthe DU145 component cell line (1,943±1,291 SFU) (p=0.704) (post-hocDunn's test for multiple comparisons). (FIG. 91C).

Antigen specific responses against ten PCa antigens was determined forthe same four donors described above for the individual modified celllines comprising PCa vaccine-A and PCa vaccine-B (Table 73. Donors 1, 2,4 and 5). IFNγ responses to TAAs induced by PCa vaccine-A and PCavaccine-B were more robust than compared to responses induced by theindividual modified PCa cell line components. Specifically, PCavaccine-A associated responses against the ten assayed antigens(9,412±6,170 SFU) were greater than responses induced by modified PC3(2,357±1,076 SFU), NEC8 (3,491±1,196 SFU) and NTERA-2c1-D1 (1,381±429SFU SFU). There was a trend towards increased IFNγ production for PCavaccine-A compared to individual modified cell lines, but this trend didnot reach statistical significance likely due to the low n of Donors(n=4) (FIG. 100D). PCa vaccine-B induced responses against the tenassayed antigens (12,067±6,694 SFU) were significantly different thanthe individual component cell lines (p=0.047, Kruskal Wallis test).Specifically, PCa vaccine-B antigen specific responses weresignificantly greater then responses those induced by modified DU145(2,064±1,604 SFU) (p=0.0345), but not LNCaP (1,419±189 SFU) (p=0.113) orDMS 53 (2,615±1,044 SFU) (p=0.544) (post-hoc Dunn's test for multiplecomparisons) (FIG. 91E). Collectively, the data described abovedemonstrate that compositions comprising a therapeutically effectiveamount of three cancer cell lines induce more robust IFNγ responses tounmodified parental cell lines and PCa antigens than a single cell linecomposition.

TABLE 73 IFNγ Responses to unmodified and modified PCa vaccinecomponents Donor Unmodified (SFU ± SEM) Modified (SFU ± SEM) (n = 4) PCavaccine-A PCa vaccine-B PCa Vaccine PCa vaccine-A PCa vaccine-B PCaVaccine 1 729 ± 243 7,608 ± 2,463 8,337 ± 2,584 251 ± 251 1,652 ± 882  3,588 ± 1,844 2 320 ± 241 1,545 ± 663   2,430 ± 841   10,603 ± 6,129 12,750 ± 8,596  30,478 ± 18,894 3 1,608 ± 360   461 ± 272 4,519 ± 1,3148,400 ± 2,027 13,863 ± 3,296  46,955 ± 10,118 4 3,781 ± 2,630 3 ± 33,784 ± 2,630 2,753 ± 630   2,749 ± 1,141 7,471 ± 2,329 5 25 ± 25 505 ±221 530 ± 243 26,323 ± 12,033 10,649 ± 6,413  42,613 ± 19,867 6 56 ± 45214 ± 93  270 ± 124 3,621 ± 1,500 16,753 ± 1,766  20,961 ± 3,534  73,028 ± 1,007 1,789 ± 561   4,824 ± 1,363 2,395 ± 1,031 4,135 ± 1,8117,399 ± 2,637

Based on the disclosure and data provided herein, a whole cell vaccinefor prostate cancer comprising the six cancer cell lines, sourced fromATCC or JCRB, PC-3 (ATCC, CRL-1435), NEC-8 (JCRB, JCRB0250),NTERA-2c1-D1 (ATCC, CRL-1973), DU145 (ATCC, HTB-81), LNCaP (ATCC,CRL-2023) and DMS 53 (ATCC, CRL-2062) is shown in Table 74. The celllines represent five prostate cancer and testicular cancer cell linesand one small cell lung cancer (SCLC) cell line (DMS 53 ATCC CRL-2062).The cell lines have been divided into two groupings: vaccine-A andvaccine-B. Vaccine-A is designed to be administered intradermally in theupper arm and vaccine-B is designed to be administered intradermally inthe thigh. Vaccine A and B together comprise a unit dose of cancervaccine.

TABLE 74 Cell line nomenclature and modifications TGFβ1 TGFβ2 CD276Cocktail Cell Line KD KD KO GM-CSF CD40L IL-12 TAA(s) A PC3 X X X X X XX A NEC8 ND ND X X X X ND A NTERA-2cl-D1 ND ND X X X X ND B DU-145 ND NDX X X X X B LNCaP ND ND X X X X ND B DMS 53* ND X X X X X ND ND = Notdone. ^(∧)CD276 KD. *Cell lines identified as CSC-like cells.

Where indicated in the above table, the genes for the immunosuppressivefactors transforming growth factor-beta 1 (TGFβ1) and transforminggrowth factor-beta 2 (TGFβ2) have been knocked down using shRNAtransduction with a lentiviral vector. The gene for CD276 has beenknocked out by electroporation using zinc-finger nuclease (ZFN) orknocked down using shRNA transduction with a lentiviral vector. Thegenes for granulocyte macrophage—colony stimulating factor (GM-CSF),IL-12, CD40L, modTBXT (PC3), modMAGEC2 (PC3), and modPSMA (DU145) havebeen added by lentiviral vector transduction.

The present Example thus provides two compositions comprising atherapeutically effective amount of three cancer cell lines each (i.e.,a unit dose of six cancer cell lines), modified to reduce the expressionof at least one immunosuppressive factor and to express at least twoimmunostimulatory factors. One composition, PCa vaccine-A, was modifiedto increase the expression of two TAAs, modTBXT and modMAGEC2. Thesecond composition, PCa vaccine-B, was modified to expresses one TAA,modPSMA. The unit dose of six cancer cell lines expresses at least atleast 18 TAAs associated with a cancer of a subset of PCa cancersubjects intended to receive said composition and induces IFNγ responses6.1-fold greater than the unmodified composition components.

Example 32: Preparation of Urinary Bladder Cancer (UBC) Vaccine

This Example demonstrates that reduction of TGFβ1, TGFβ2, and CD276expression with concurrent overexpression of GM-CSF, CD40L, and IL-12 ina vaccine composition of two cocktails, each cocktail composed of threecell lines for a total of 6 cell lines, significantly increased themagnitude of cellular immune responses to at least 10 UBC-associatedantigens in an HLA-diverse population. As described herein, the firstcocktail, UBC vaccine-A, is composed of cell line J82 that was alsomodified to express modPSMA and modCriptol (modTDGF1), cell lineHT-1376, and cell line TCCSUP. The second cocktail, UBC vaccine-B, iscomposed of cell line SCaBER that was also modified to express modWT1and modFOLR1 (modFBP), cell line UM-UC-3, and cell line DMS 53. The sixcomponent cell lines collectively express at least twenty-four antigensthat can provide an anti-UBC tumor response.

Identification of UBC Vaccine Components

Initial cell line selection criteria identified twenty-six vaccinecomponent cell lines for potential inclusion in the UBC vaccine.Additional selection criteria described herein were applied to narrowthe twenty-six cell lines to eight cell lines for further evaluation inimmunogenicity assays. These criteria included: endogenous UBCassociated antigen expression, lack of expression of additionalimmunosuppressive factors, such as IL-10 or IDO1, expression ofUBC-associated CSC-like markers YAP1, ALDH1A, CD44, CEACAM6, and Oct4,ethnicity and age of the patient from which the cell line was derived,site and stage of the bladder cancer, and histological subtype.

CSCs play a critical role in the metastasis, treatment resistance, andrelapse of bladder cancer (Table 2). Expression of TAAs and UBC specificCSC-like markers by candidate component cell lines was determined by RNAexpression data sourced from the Broad Institute Cancer Cell LineEncyclopedia (CCLE). The HGNC gene symbol was included in the CCLEsearch and mRNA expression was downloaded for each TAA. Expression of aTAA or CSC marker by a cell line was considered positive if the RNA-seqvalue was greater than one. Selection criteria identified eightcandidate UBC vaccine components for further evaluation: UM-UC-3, J82,T24, HT-1376, HT-1197, TCCSUP, SCaBER, and RT-4. The eight candidatecomponent cell lines expressed nine to seventeen TAAs (FIG. 92A) and twoor three CSC markers (FIG. 92B). As described herein, the CSC-like cellline DMS 53 is included as one of the six vaccine cell lines andexpressed fifteen UBC TAAs and three UBC CSC-like markers.

Immunogenicity of the eight unmodified UBC vaccine component candidateswas evaluated by IFNγ ELISpot as described in Example 9 using three HLAdiverse healthy donors (n=4 per donor). HLA-A and HLA-B alleles forDonor 1 were A*02:01 B*35:02 and A*02:01 B*49:01. HLA-A and HLA-Balleles for Donor 2 were A*32:01 B*27:05 and A*68:05 B*39:08. HLA-Aalleles for Donor 3 were A*01:01 and A*03:01. HLA-B typing was notavailable for Donor 3. J82 (5,420±577 SFU), TCCSUP (3,504±702 SFU) andSCaBER (2,903±654 SFU) were more immunogenic than UM-UC-3 (1,022±284SFU), T24 (1,492±211 SFU), HT-1376 (922±230 SFU), HT-1197 (63±63 SFU)and RT-4 (13±13 SFU) (FIG. 93A).

Immunogenicity of J82 and TCCSUP was evaluated in eight differentcombinations of three component cell lines, four combinations containedJ82 and four combinations contained TCCSUP (FIG. 93C). IFNγ responseswere determined against the three component cell lines within in theeight potential vaccine cocktails by IFNγ ELISpot as described inExample 8 using the three healthy donors (n=4/donor). HLA-A and HLA-Balleles for Donor 1 were A*01:01 B*08:01 and A*02:01 B*15:01. HLA-A andHLA-B alleles for Donor 2 were A*03:01 B*15:01 and A*24:02 B*07:02.HLA-typing was only available for one HLA-A allele for Donor 3, whichwas A*02:01. Donor 3 HLA-B alleles were B*15:01 and B*51:01. IFNγresponses were detected for all eight cocktails and to each cell linecomponent in each cocktail. Responses to the individual cocktailcomponent cell lines were notably decreased compared to IFNγ responsesdetected for single cell line components (FIG. 93B). In all eightcombinations evaluated, TCCSUP remained the most immunogenic. HT-1197was poorly immunogenic alone and in three cell line component cocktailsand therefore not included in the UBC vaccine. The immunogenicity ofJ82, T24 and SCaBER was similar when evaluated in three cell linecomponent cocktails. Of these three cell lines, T24 endogenouslyexpressed the least number of TAAs (nine TAAs>1.0 FPKM) (FIG. 92A) andwas excluded from the UBC vaccine. J82 and SCaBER were selected toexpress UBC antigens by lentiviral transduction as described above andplaced in separate vaccine cocktails to mitigate any potential forantigen competition when delivered in the same vaccine cocktail. TCCSUPand J82 were selected to be included in vaccine cocktail A and SCaBERselected to be included in vaccine cocktail B as described above andfurther herein.

The cells in the vaccine described herein were selected to express awide array of TAAs, including those known to be important specificallyfor UBC antitumor responses, such as Criptol or DEPDC1, and also TAAsknown to be important for targets for UBC and other solid tumors, suchTERT. As shown herein, to further enhance the array of TAAs, J82 wasmodified to express modPSMA and modCriptol (TDGF1) and SCaBER wasmodified to express modWT1 and modFOLR1 (FBP). Criptol (TDGF1) was notendogenously expressed in any of the six component cell lines at >1.0FPKM. PSMA, FOLR1 (FBP) and WT1 were endogenously expressed by one ofthe six component cell lines at >1.0 FPKM (FIG. 94A).

Expression of the transduced antigens modPSMA (FIG. 95A) and modCriptol(modTDGF1) (FIG. 95B) by J82 (SEQ ID NO: 53; SEQ ID NO: 54), and modWT1(FIG. 95C) and modFOLR1 (modFBP) (FIG. 94D) (SEQ ID NO: 51; SEQ ID NO:52) by SCaBER, were detected by flow cytometry or RT-PCR as described inExample 29 and herein. The modPSMA and Criptol (TDGF1) antigens areencoded in the same lentiviral transfer vector separated by a furincleavage site (SEQ ID NO: 53; SEQ ID NO: 54). The modWT1 and modFOLR1(FBP) are encoded in the same lentiviral transfer vector separated by afurin cleavage site (SEQ ID NO: 52).

Because of the need to maintain maximal heterogeneity of antigens andclonal subpopulations the comprise each cell line, the gene modifiedcell lines utilized in the present vaccine have been established usingantibiotic selection and flow cytometry and not through limitingdilution subcloning.

The endogenous mRNA expression of twenty-four representative UBC TAAs inthe present vaccine are shown in FIG. 94A. The present vaccine, afterintroduction antigens described above, expresses of all identifiedtwenty-four commonly targeted and potentially clinically relevant TAAscapable of inducing a UBC antitumor response. Some of these TAAs areknown to be primarily enriched in UBC tumors and some can also induce animmune response to UBC and other solid tumors. RNA abundance of thetwenty-four prioritized UBC TAAs was determined in 407 UBC patientsamples with available mRNA data expression as described in Example 29(FIG. 94B). Fifteen of the prioritized UBC TAAs were expressed by 100%of samples, 16 TAAs were expressed by 99.3% of samples, 17 TAAs wereexpressed by 96.8% of samples, 18 TAAs were expressed by 90.7% ofsamples, 19 TAAs were expressed by 80.3% of samples, 20 TAAs wereexpressed by 68.6% of samples, 21 TAAs were expressed by 56.3% ofsamples, 22 TAAs were expressed by 41.3% of samples, 23 TAAs wereexpressed by 27.5% of samples and 24 TAAs were expressed by 9.1% ofsamples (FIG. 94C). Thus, provided herein are two compositionscomprising a therapeutically effective amount of three cancer celllines, wherein the combination of the cell lines, a unit dose of sixcell lines, comprises cells that express at least 15 TAAs associatedwith a subset of UBC cancer subjects intended to receive saidcomposition. Based on the expression and immunogenicity data presentedherein, the cell lines identified in Table 75 were selected to comprisethe present UBC vaccine.

TABLE 75 Bladder vaccine cell lines and histology Cocktail Cell LineName Histology A J82 Bladder Transitional Cell Carcinoma A HT-1376Bladder Grade III Carcinoma A TCCSUP Bladder Anaplastic Grade IVTransitional Cell Carcinoma B SCaBER Bladder Squamous Cell Carcinoma BUM-UC-3 Bladder Transitional Cell Carcinoma B DMS 53 Lung Small CellCarcinoma

Reduction of CD276 Expression

The J82, HT-1376, TCCSUP, SCaBER, UM-UC-3 and DMS 53 component celllines expressed CD276 and expression was knocked out by electroporationwith ZFN as described in Example 13 and elsewhere herein. Because it wasdesirable to maintain as much tumor heterogeneity as possible, theelectroporated and shRNA modified cells were not cloned by limitingdilution. Instead, the cells were subjected to multiple rounds of cellsorting by FACS as described in Example 13. Expression of CD276 wasdetermined as described in Example 29. Reduction of CD276 expression isdescribed in Table 76. These data show that gene editing of CD276 withZFN resulted in greater than 99.8% CD276-negative cells in all sixvaccine component cell lines.

TABLE 76 Reduction of CD276 expression Parental Cell Modified Cell %Reduction Cell line Line MFI Line MFI CD276 J82 13,721 27 99.8 HT-137627,871  0 >99.9 TCCSUP 21,401 37 99.8 SCaBER 31,950 29 99.9 UM-UC-3 2,135  2 99.9 DMS 53 11,928 24 99.8 MFI reported with isotype controlssubtracted

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12 werecompleted as described in Example 29.

shRNA Downregulates TGF-β Secretion

Following CD276 knockout, TGFβ1 and TGFβ2 secretion levels were reducedusing shRNA and resulting levels determined as described in Example 29.The J82, HT-1376 and TCCSUP parental cell lines in UBC vaccine-Asecreted measurable levels of TGFβ1 and TGFβ2. J82 secreted low levelsof TGFβ1 and was not modified to reduce TGFβ1 secretion. The SCaBER andUM-UC-3 component cell lines of UBC vaccine-B secreted measurable levelsof TGFβ1. SCaBER also secreted measurable levels of TGFβ2. Reduction ofTGFβ2 secretion by the DMS 53 cell line is described in Example 26 andresulting levels determined as described above and herein.

The HT-1376, TCCSUP, SCaBER component cell lines were transduced withTGFβ1 shRNA to decrease TGFβ1 secretion concurrently with the transgeneto increase expression of membrane bound CD40L as described in Example29. HT-1376, TCCSUP, SCaBER were also transduced with lentiviralparticles encoding TGFβ2 shRNA to decrease the secretion of TGFβ2 andconcurrently increase expression of GM-CSF (SEQ ID NO: 6) as describedin Example 29. These cells are described by the clonal designation DK6.The UM-UC-3 cell line was transduced with TGFβ1 shRNA to decrease TGFβ1secretion and concurrently increase expression of membrane bound CD40Las described in Example 29. These cells, modified to reduce TGFβ1secretion and not TGFβ2 secretion, are described by the clonaldesignation DK2. J82 was transduced with lentiviral particles encodingTGFβ2 shRNA to decrease the secretion of TGFβ2 and concurrently increaseexpression of GM-CSF (SEQ ID NO: 6) as described in Example 29. DMS 53was modified with shRNA to reduce secretion of TGFβ2 as described inExample 26. The J82 and DMS 53 cells modified to reduce secretion ofTGFβ2 and not TGFβ1 are described by the clonal designation DK4.

Table 77 shows the percent reduction in TGFβ1 and/or TGFβ2 secretion ingene modified component cell lines compared to unmodified, parental,cell lines. Gene modification resulted in at least 78% reduction ofTGFβ1 secretion. Gene modification of TGFβ2 resulted in at least 51%reduction in secretion of TGFβ2.

TABLE 77 TGF-β Secretion (pg/10⁶ cells/24 hr) in Component Cell LinesCell Line Cocktail Clone TGFβ1 TGFβ2 J82 A Wild type * ≤24 955 ± 462 J82A DK4 NA  * ≤8 J82 A Percent reduction NA ≥99% HT-1376 A Wild type 817 ±206 230 ± 86  HT-1376 A DK6 * ≤49 * ≤23 HT-1376 A Percent reduction ≥94%≥90% TCCSUP A Wild type 2,273 ± 502   675 ± 157  TCCSUP A DK6 133 ± 26 62 ± 24 TCCSUP A Percent reduction  94%  91% SCaBER B Wild type 85 ± 131,954 ± 341   SCaBER B DK6 * ≤18 224 ± 35  SCaBER B Percent reduction 79%  89% UM-UC-3 B Wild type 375 ± 80   * ≤8 UM-UC-3 B DK2 81 ± 12 NAUM-UC-3 B Percent reduction  78% NA DMS 53 B Wild type 106 ± 10  486 ±35  DMS 53 B DK4 NA 238 ± 40  DMS 53 B Percent reduction NA  51% DK6:TGFβ1/TGFβ2 double knockdown; DK4: TGFβ2 single knockdown; DK2: TGFβ1single knockdown; * = estimated using LLD, not detected; NA = notapplicable

Based on a dose of 5×10⁵ of each component cell line, the total TGFβ1and TGFβ2 secretion by the modified UBC vaccine-A and UBC vaccine-B andrespective unmodified parental cell lines are shown in Table 78. Thesecretion of TGFβ1 by UBC vaccine-A was reduced by 93% pg/dose/24 hr andTGFβ2 by 95% pg/dose/24 hr. The secretion of TGFβ1 by UBC vaccine-B wasreduced by 64% pg/dose/24 hr and TGFβ2 by 81% pg/dose/24 hr.

TABLE 78 Total TGF-β Secretion (pg/dose/24 hr) in UBC vaccine-A and UBCvaccine-B Cocktail Clones TGFβ1 TGFβ2 A Wild type 1,557   930 DK/DK6  103    47 Percent reduction 93% 95% B Wild type   283 1,224DK2/DK4/DK6   103 235 Percent reduction 64% 81%

GM-CSF Secretion

The HT-1376, TCCSUP, SCaBER and J82 cell lines were transduced withlentiviral particles containing both TGFβ2 shRNA and the gene to expressGM-CSF (SEQ ID NO: 6) as described above. The UM-UC-3 cell line wastransduced with lentiviral particles to only express GM-CSF (SEQ ID NO:7). DMS 53 was modified to secrete GM-CSF as described in Example 24 andelsewhere herein. The results are shown in Table 79 and described below.

Secretion of GM-CSF increased at least 2,700-fold in all modifiedcomponent cell lines compared to unmodified, parental cell lines. Foldincrease in expression of GM-CSF by the UBC vaccine-A component celllines was as follows: J82 increased 2,700-fold relative to theunmodified cell line (≤0.010 ng/10⁶ cells/24 hr); HT-1376 increased6,500-fold relative to the unmodified cell line (≤0.030 ng/10⁶ cells/24hr); TCCSUP increased 2,500-fold relative to the unmodified cell line(≤0.012 ng/10⁶ cells/24 hr). Fold increase in expression of GM-CSF bythe UBC vaccine-B component cell lines was as follows: SCaBER increased12,556-fold relative to the unmodified cell line (≤0.009 ng/10⁶ cells/24hr); UM-UC-3 increased 15,500-fold relative to the unmodified cell line(≤0.008 ng/10⁶ cells/24 hr); DMS 53 increased 39,450-fold relative tothe unmodified cell line (≤0.004 ng/10⁶ cells/24 hr).

TABLE 79 GM-CSF Secretion in Component Cell Lines GM-CSF GM-CSF CellLine (ng/10⁶ cells/24 hr) (ng/dose/24 hr) J82 27 ± 8  14 HT-1376 195 ±59  98 TCCSUP 30 ± 9  15 Cocktail A Total 252 127  SCaBER 113 ± 30  57UM-UC-3 124 ± 35  62 DMS 53 158 ± 15  79 Cocktail B Total 395 198 

Based on a dose of 5×10⁵ of each component cell line, the total GM-CSFsecretion for UBC vaccine-A was 127 ng per dose per 24 hours. The totalGM-CSF secretion for UBC vaccine-B was 198 ng per dose per 24 hours. Thetotal GM-CSF secretion per dose was therefore 325 ng per 24 hours.

Membrane Bound CD40L (CD154) Expression

The component cell lines were transduced with lentiviral particles toexpress membrane bound CD40L vector as described above. The methods todetect expression of CD40L by the five UBC cell line components aredescribed in Example 29. Modification of DMS 53 to express membranebound CD40L is described in Example 15. Evaluation of membrane boundCD40L by all six vaccine component cell lines is described below. Theresults shown in FIG. 96 and described below demonstrate CD40L membraneexpression was substantially increased in all six UBC vaccine componentcell lines.

Expression of membrane bound CD40L increased at least 851-fold in allcomponent cell lines compared to unmodified, parental cell lines. In UBCvaccine-A component cell lines expression of CD40L increased 37,196-foldby J82 (37,196 MFI) compared to the parental cell line (0 MFI), 851-foldby HT-1376 (37,444 MFI) compared to the parental cell line (44 MFI), and1,062-fold by TCCSUP (199,687 MFI) compared to the parental cell line(188 MFI). In UBC vaccine-B component cell lines expression of CD40Lincreased 13,772-fold by SCaBER (13,772 MFI) compared to the parentalcell line (0 MFI), 11,301-fold by UM-UC-3 (11,301 MFI) compared to theparental cell line (0 MFI), and 88,261-fold by DMS 53 compared to theparental cell line (0 MFI).

IL-12 Expression

The component cell lines were transduced with the IL-12 vector asdescribed in Example 17 and resulting IL-12 p70 expression determined asdescribed above and herein. The results are shown in Table 80 anddescribed below.

Secretion of IL-12 increased at least 1,400-fold in all component celllines modified to secrete IL-12 p70 compared to unmodified, parentalcell lines. In UBC vaccine-A component cell lines, secretion of IL-12increased 3,500-fold by J82 compared to the parental cell line (≤0.004ng/10⁶ cells/24 hr), 609,000-fold by HT-1376 compared to the parentalcell line (≤0.001 ng/10⁶ cells/24 hr), and 1,400-fold by TCCSUP comparedto the parental cell line (≤0.005 ng/10⁶ cells/24 hr). In UBC vaccine-Bcomponent cell lines expression of IL-12 increased 6,750-fold by SCaBERcompared to the parental cell line (≤0.004 ng/10⁶ cells/24 hr) and6,000-fold by UM-UC-3 compared to the parental cell line (≤0.003 ng/10⁶cells/24 hr). DMS 53 was not modified to secrete IL-12.

TABLE 80 IL-12 Secretion in Component Cell Lines IL-12 IL-12 Cell Line(ng/10⁶ cells/24 hr) (ng/dose/24 hr) J82 14 ± 4    7 HT-1376 609 ± 51 305 TCCSUP 7 ± 3   4 Cocktail A Total 630 316 SCaBER 27 ± 12  14 UM-UC-318 ± 19   9 DMS 53 NA NA Cocktail B Total  45  23

Based on a dose of 5×10⁵ of each component cell line, the total IL-12secretion for UBC vaccine-A was 316 ng per dose per 24 hours. The totalIL-12 secretion for UBC vaccine-B was 23 ng per dose per 24 hours. Thetotal IL-12 secretion per dose was therefore 339 ng per 24 hours.

Stable Expression of modPSMA and modCripto1(modTDGF1) by the J82 CellLine

As described above, the cells in the vaccine described herein wereselected to express a wide array of TAAs, including those known to beimportant to antitumor immunity. To further enhance the array ofantigens, the J82 cell line that was modified to reduce the secretion ofTGFβ2, reduce the expression of CD276, and to express GM-CSF, membranebound CD40L and IL-12 was also transduced with lentiviral particlesexpressing the modPSMA and modCriptol antigens. The genes encoding themodPSMA and modCriptol antigens are linked by a furin cleavage site (SEQID NO: 53, SEQ ID NO: 54).

The expression of modPSMA by J82 was characterized by flow cytometry.Unmodified and antigen modified cells were stained intracellular with0.03 μg/test anti-mouse IgG1 anti-PSMA antibody (Abcam, ab268061)followed by 0.125 ug/test AF647-conjugated goat anti-mouse IgG1 antibody(BioLegend #405322). Expression of modPSMA was increased in the modifiedcell line (249,632 MFI) 60-fold over that of the parental cell line(16,481 MFI) (FIG. 95A). Expression of modCriptol by J82 was alsocharacterized by flow cytometry. Cells were first stained intracellularwith rabbit IgG anti-Criptol antibody (Abcam, ab108391) (0.03 μg/test)followed by AF647-conjugated donkey anti-rabbit IgG1 antibody (BioLegend#406414) (0.125 μg/test). Expression of modCriptol increased in themodified cell line (3,330,400 MFI) 255-fold over the unmodified cellline (13,042 MFI) (FIG. 94B).

Stable Expression of modWT1 and modFOLR1 (modFBP) by the SCaBER CellLine

The SCaBER cell line that was modified to reduce the secretion of TGFβ1and TGFβ2, reduce the expression of CD276, and to express GM-CSF,membrane bound CD40L, and IL-12 was also transduced with lentiviralparticles expressing the modWT1 and modFOLR1 antigens (SEQ ID NO: 51,SEQ ID NO: 52). Expression of modWT1 by SCaBER was characterized by flowcytometry. Unmodified and antigen modified cells were stainedintracellular with 0.03 μg/test anti-rabbit IgG1 anti-WT1 antibody(Abcam, ab89901) followed by 0.125 ug/test AF647-conjugated donkeyanti-rabbit IgG1 antibody (BioLegend #406414). Expression of modWT1increased in the modified cell line (4,121,028 MFI) 90-fold over that ofthe unmodified cell line (46,012 MFI) (FIG. 94C). Expression of modFOLR1by SCaBER was determined by RT-PCR as described in Example 29 andherein. The forward primer was designed to anneal at the 56-76 bplocation in the transgene (GAGAAGTGCAGACCAGAATCG (SEQ ID NO: 130)) andreverse primer designed to anneal at the 588-609 bp location in thetransgene (TCTGCTGTAGTTGGACACCTTG (SEQ ID NO: 131)) yielding a 554 bpproduct. Control primers for β-tubulin are described in Example 29. Thegene product for modFOLR1 was detected at the expected size (FIG. 95D)and mRNA increased 249,810-fold relative to the parental control.

Immune Responses to PSMA and Criptol (TDGF1) in UBC Vaccine-A

IFNγ responses to PSMA and Criptol were evaluated in the context of UBCvaccine-A as described in Example 29, and herein, in seven HLA diversedonors (n=4/donor). The HLA-A, HLA-B, and HLA-C alleles for each of theseven donors are shown in Table 81. IFNγ responses were determined byELISpot as described in Example 29.

PSMA specific IFNγ responses with the were increased with the modifiedUBC vaccine-A (757±278 SFU) compared to the parental, unmodified UBCvaccine-A (450±179 SFU (FIG. 95E). IFNγ responses to Criptol weredetermined by ELISpot using 15-mers peptides overlapping by 9 aminoacids spanning the entire length of the native Criptol antigen purchasedfrom Thermo Scientific Custom Peptide Service. IFNγ responses to Criptolsignificantly increased with the modified UBC vaccine-A (420±132 SFU)compared to the unmodified UBC vaccine-A (67±47 SFU) (p=0.023,Mann-Whitney U test) (n=7) (FIG. 95F).

Immune Responses to WT1 and FOLR1 (FBP) in UBC Vaccine-B

IFNγ responses to WT1 and FOLR1 were evaluated in the context ofUBC-vaccine B as described in Example 29, and herein, in seven HLAdiverse donors (n=4/donor) (Table 81). IFNγ responses against WT1 andFOLR1 were determined by ELISpot using 15-mers peptides overlapping by 9amino acids spanning the entire length of the native antigen proteinpurchased from Thermo Scientific Custom Peptide Service. WT1 specificIFNγ responses were significantly increased by UBC vaccine-B (654±268SFU) compared to the unmodified UBC vaccine-B (65±23 SFU) (p=0.017,Mann-Whitney U test) (n=7) (FIG. 95G). FOLR1 specific IFNγ responseswere significantly increased by UBC vaccine-B (643±244 SFU) compared tothe unmodified UBC vaccine-B (95±51 SFU) (p=0.011, Mann-Whitney U test)(n=7) (FIG. 95H).

TABLE 81 Healthy Donor MHC-I characteristics Donor # HLA-A HLA-B HLA-C 1*02:01 *11:01 *07:02 *37:02 *06:02 *07:02 2 *03:01 *03:01 *07:02 *18:01*07:02 *12:03 3 *02:01 *02:01 *15:01 *51:01 *02:02 *03:04 4 *01:01*30:01 *08:01 *13:02 *06:02 *07:02 5 *02:01 *30:02 *14:02 *13:02 *08:02*18:02 6 *03:01 *32:01 *07:02 *15:17 *07:01 *07:02 7 *02:01 *25:01*18:01 *27:05 *02:02 *12:03

Cocktails Induce Immune Responses Against Relevant TAAs

The ability of UBC vaccine-A and UBC vaccine-B to induce IFNγ productionagainst ten UBC antigens was measured by ELISpot. PBMCs from sevenHLA-diverse healthy donors (Table 81) were co-cultured with autologousDCs loaded with UBC vaccine-A or UBC vaccine-B for 6 days prior tostimulation with TAA-specific specific peptide pools containing knownMHC-I restricted epitopes. Peptides for stimulation of CD14-PBMCs todetect IFNγ responses to PSMA, Criptol, WT1 and FOLR1 are describedabove. Additional 15-mer peptides overlapping by 11 amino acid peptidepools were sourced as follows: Survivin (thinkpeptides, 7769_001-011),MUC1 (JPT, PM-MUC1), MAGEA1 (JPT, PM-MAGEA1), MAGEA3 (JPT, PM-MAGEA3),TERT (JPT, PM-TERT) and STEAP1 (PM-STEAP1).

FIG. 97 demonstrates the UBC vaccine is capable of inducing antigenspecific IFNγ responses in seven HLA-diverse donors to ten UBC antigensthat are 4.3-fold more robust (12,706±3,223 SFU) compared to theunmodified parental control (2,986±813 SFU) (p=0.007, Mann-Whitney Utest) (n=7) (FIG. 97A) (Table 82). The unit dose of UBC vaccine-A andUBC vaccine-B elicited IFNγ responses to eight antigens in two donors,nine antigens in one donor and ten antigens in four donors (FIG. 98).UBC vaccine-A and UBC vaccine-B independently demonstrated a 2.5-foldand 7.9-fold increase antigen specific responses compared to parentalcontrols, respectively. Specifically, UBC vaccine-A elicited 5,140±1,422SFU compared to the unmodified controls (2,027±573 SFU) (FIG. 97B). ForUBC vaccine-A, one donor responded to four antigens, one donor respondedto six antigens, one donor responded to seven antigens, one donorresponded to seven antigens, and three donors responded ten antigens.UBC vaccine-B elicited 7,565±1,933 SFU compared to parental controls(959±331 SFU) (p=0.011, Mann-Whitney U test) (FIG. 97C). For UBCvaccine-B, one donor responded to four antigens, one donor responded toeight antigens, one donor responded to nine antigens, and four donorsresponded to ten antigens. Described above are two compositionscomprising a therapeutically effective amount of three cancer celllines, a unit dose of six cell lines, wherein said unit dose is capableof eliciting an immune response 4.3-fold greater than the unmodifiedcomposition specific to at least eight TAAs expressed in UBC patienttumors. UBC vaccine-A increased IFNγ responses to at least four TAAs2.5-fold and UBC vaccine-B increased IFNγ responses 7.9-fold to at leastfour TAAs.

TABLE 82 IFNγ Responses to unmodified and modified UBC vaccinecomponents Donor Unmodified (SFU ± SEM) Modified (SFU ± SEM) (n = 4) UBCvaccine-A UBC vaccine-B UBC Vaccine UBC vaccine-A UBC vaccine-B UBCVaccine 1 319 ± 71  415 ± 18  734 ± 78  2,058 ± 1,247 6,667 ± 4,4598,725 ± 5,658 2 3,568 ± 268   2,905 ± 300   6,473 ± 128   9,138 ± 2,36315,225 ± 1,123  24,363 ± 3,099  3 3,270 ± 1,234 845 ± 339 4,115 ± 1,0221,549 ± 343   5,376 ± 1,730 6,924 ± 1,986 4 3,141 ± 715   841 ± 5273,982 ± 788   9,881 ± 1,359 13,551 ± 1,749  23,432 ± 2,220  5 318 ± 183405 ± 268 723 ± 440 1,100 ± 902   551 ± 551 1,651 ± 1,452 6* 2,945 ±816   614 ± 406 3,559 ± 1,031 7,838 ± 3,795 6,603 ± 3,431 14,440 ±7,091  7 628 ± 146 688 ± 193 1,315 ± 327   4,420 ± 1,896 4,985 ± 1,7259,405 ± 3,522 *Donor 6, n = 3. All other donors, n = 4.

Based on the disclosure and data provided herein, a whole cell vaccinefor Bladder Cancer comprising the six cancer cell lines, sourced fromATCC, J82 (ATCC, HTB-1), HT-1376 (ATCC, CRL-1472), TCCSUP (ATCC, HTB-5),SCaBER (ATCC, HTB-3), UM-UC-3 (ATCC, CRL-1749) and DMS 53 (ATCC,CRL-2062) is shown in Table 83. The cell lines represent five bladdercancer cell lines and one small cell lung cancer (SCLC) cell line (DMS53 ATCC CRL-2062). The cell lines have been divided into two groupings:vaccine-A and vaccine-B. Vaccine-A is designed to be administeredintradermally in the upper arm and vaccine-B is designed to beadministered intradermally in the thigh. Vaccine A and B togethercomprise a unit dose of cancer vaccine.

TABLE 83 Cell line nomenclature and modifications TGFβ1 TGFβ2 CD276Cocktail Cell Line KD KD KO GM-CSF CD40L IL-12 TAA(s) A J82 ND X X X X XX A HT-1376 X X X X X X ND A TCCSUP X X X X X X ND B SCaBER X X X X X XX B UM-UC-3 X ND X X X X ND B DMS 53* ND X X X X X ND ND = Not done.*Cell lines identified as CSC-like cells.

Where indicated in the above table, the genes for the immunosuppressivefactors transforming growth factor-beta 1 (TGFβ1) and transforminggrowth factor-beta 2 (TGFβ2) have been knocked down using shRNAtransduction with a lentiviral vector. The gene for CD276 has beenknocked out by electroporation using zinc-finger nuclease (ZFN) orknocked down using shRNA transduction with a lentiviral vector. Thegenes for granulocyte macrophage—colony stimulating factor (GM-CSF),IL-12, CD40L, modPSMA (J82), modCriptol (modTDGF1) (J82), modWT1(SCaBER) and modFOLR1 (modFBP) (SCaBER) have been added by lentiviralvector transduction.

The present Example thus provides re two compositions comprising atherapeutically effective amount of three cancer cell lines, a unit doseof six cancer cell lines, modified to reduce the expression of at leasttwo immunosuppressive factors and to express at least twoimmunostimulatory factors. One composition, UBC vaccine-A, was modifiedto increase the expression of two TAAs, modPSMA and modCriptol(modTDGF1). The second composition, UBC vaccine-B, was modified toexpresses two TAAs, modWT1 and modFOLR1 (modFBP). The unit dose of sixcancer cell lines expresses at least at least 15 TAAs associated with acancer of a subset of bladder cancer subjects intended to receive saidcomposition and induces IFNγ responses 4.3-fold greater than theunmodified composition components.

Example 33: Preparation of Ovarian Cancer (OC) Vaccine

This Example demonstrates that reduction of TGFβ1, TGFβ2, and CD276expression with concurrent overexpression of GM-CSF, CD40L, and IL-12 ina vaccine composition of two cocktails, each cocktail composed of threecell lines for a total of 6 cell lines, significantly increased themagnitude of cellular immune responses to at least 10 OC-associatedantigens in an HLA-diverse population. As described herein, the firstcocktail, OC vaccine-A, is composed of cell line OVTOKO, cell line MCASthat was also modified to express modTERT, and cell line TOV-112D thatwas also modified to express modFSHR and modMAGEA10. The secondcocktail, OC vaccine-B, is composed of cell line TOV-21G that was alsomodified to express modWT1 and modFOLR1 (modFBP), cell line ES-2 thatwas also modified to express modBORIS, and cell line DMS 53. The sixcomponent cell lines collectively express at least twenty antigens thatcan provide an anti-OC tumor response.

Identification of OC Vaccine Components

Initial cell line selection criteria identified thirty-six vaccinecomponent cell lines for potential inclusion in the OC vaccine.Additional selection criteria described herein were applied to narrowthe thirty-six cell lines to ten cell lines for further evaluation inimmunogenicity assays. These criteria included: endogenous OC associatedantigen expression, lack of expression of additional immunosuppressivefactors, such as IL-10 or IDO1, expression of OC-associated CSC-likemarkers ALDH1A, EPCAM, CD44, CD133, CD117, Endoglin, Oct4, NANOG andSAL4, ethnicity and age of the patient from which the cell line wasderived, if the cell line was derived from a primary tumor or metastaticsite, and ovarian histological subtype.

CSCs play a critical role in the metastasis, treatment resistance, andrelapse of ovarian cancer (Table 2). Expression of TAAs and CSC-likemarkers by candidate component cell lines was determined by RNAexpression data sourced from the Broad Institute Cancer Cell LineEncyclopedia (CCLE). The HGNC gene symbol was included in the CCLEsearch and mRNA expression was downloaded for each TAA or CSC marker.Expression of a TAA or CSC marker by a cell line was considered positiveif the RNA-seq value was greater than one. Selection criteria identifiedten candidate OC vaccine components for further evaluation: OVCAR-3,KURAMOCHI, MCAS, TYK-nu, OVSAHO, OVTOKO, TOV-21G, ES-2, OVMANA, andTOV-112D. The ten candidate component cell lines expressed six tofourteen TAAs (FIG. 99A) and two to five CSC-like markers (FIG. 99B). Asdescribed herein, the CSC-like cell line DMS 53 is included as one ofthe six vaccine cell lines and expressed twelve OC TAAs and five OCCSC-like markers.

Immunogenicity of the ten unmodified OC vaccine component candidates wasevaluated by IFNγ ELISpot as described in Example 9 for three HLAdiverse healthy donors (n=4 per donor). HLA-A and HLA-B alleles for thethree Donors were as follows: Donor 1, A*02:01 B*35:01 and A*31:01B*35:03; Donor 2, A*01:01 B*07:02 and A*30:01 B*12:02; Donor 3, A*02:01B*15:07 and A*24:02 B*18:01. KURAMOCHI (1,896±421 SFU), OVTOKO(2,124±591 SFU) and TOV-21G (1,559±273 SFU) were more immunogenic thanOVCAR-3 (54±24 SFU), MCAS (420±218 SFU), TYK-nu (339±109 SFU), OVSAHO(404±163 SFU), ES-2 (215±117 SFU), OVMANA (46±29) and TOV-112D (89±62)(FIG. 100A).

Immunogenicity of KURAMOCHI, OVTOKO and TOV-21G was evaluated in elevendifferent combinations of three component cell lines, three combinationscontained KURAMOCHI, four combinations contained OVTOKO and fourcombinations contained TOV-21G (FIG. 100C). OVMANA (JCRB, JCRB1045) wasnot included in the eleven cocktails due to poor viabilitypost-cryopreservation noted by JCRB that was confirmed prior tocompletion of the experiments described herein. IFNγ responses weredetermined against three component cell lines in the eleven potentialvaccine cocktails by IFNγ ELISpot as described in Example 8 for threehealthy donors (n=4/donor). HLA-A and HLA-B alleles for the Donors wereas follows: Donor 1, A*02:01 B*07:02 and A*23:01 B*14:02; Donor 2,A*32:01 B*27:05 and A*68:05 B*39:08; Donor 3, A*02:02 B*15:03 andA*30:02 B*57:03. IFNγ responses were detected for all eleven cocktailsand to each cell line component in each cocktail. IFNγ responses againstmost cocktail component cell lines were similar or notably increasedcompared to responses detected for single cell lines. In all elevencombinations evaluated, KURAMOCHI, OVTOKO and TOV-21G remained the mostimmunogenic (FIG. 100B). KURAMOCHI was not selected for inclusion in thefinal OC vaccine due to potential large-scale manufacturing concernsbased on growth morphology following genetic modifications. OVTOKO andTOV-21G were selected to be included in vaccine cocktail A and vaccinecocktail B, respectively, as described further herein.

The cells in the vaccine described herein were selected to express awide array of TAAs, including those known to be important specificallyfor OC antitumor responses, such as FOLR1 or FSHR, and also TAAs knownto be important for targets for OC and other solid tumors, such TERT.

As shown herein, to further enhance the array of TAAs, MCAS was modifiedto express modTERT, TOV-112D was modified to express modFSHR andmodMAGEA10, TOV-21G was modified to express modWT1 and modFOLR1 (modFBP)and ES-2 was modified to express modBORIS. FSHR, MAGEA10, WT1, FOLR1 andBORIS were not endogenously expressed in the six component cell linesat >1.0 FPKM. TERT was endogenously expressed by two of the sixcomponent cell lines at >1.0 FPKM (FIG. 101A).

Expression of the transduced antigens modTERT (FIG. 102A) (SEQ ID NO:35; SEQ ID NO: 36) by MCAS, modFSHR (FIG. 112B) and modMAGEA10 (FIG.102C) (SEQ ID NO: 43; SEQ ID NO: 44) by TOV-112D, modWT1 (FIG. 102D) andmodFOLR1 (modFBP) (FIG. 102E) (SEQ ID NO: 51; SEQ ID NO: 52) by TOV-21Gand modBORIS (FIG. 102F) (SEQ ID NO: 59; SEQ ID NO: 60) by ES-2 weredetected by flow cytometry or RT-PCR as described in Example 29 andherein. modFSHR and modMAGEA10 were encoded in the same lentiviraltransfer vector separated by a furin cleavage site. modWT1 and modFOLR1were also encoded in the same lentiviral transfer vector separated by afurin cleavage site.

Because of the need to maintain maximal heterogeneity of antigens andclonal subpopulations the comprise each cell line, the gene modifiedcell lines utilized in the present vaccine have been established usingantibiotic selection and flow cytometry and not through limitingdilution subcloning.

The endogenous mRNA expression of twenty representative OC TAAs in thepresent vaccine are shown in FIG. 101A. The present vaccine, afterintroduction of antigens described above, expresses all identifiedtwenty commonly targeted or potentially clinically relevant TAAs capableof inducing an OC antitumor response. Some of these TAAs are known to beprimarily enriched in OC tumors, such as FOLR1(FBP) or FSHR, and somecan also induce an immune response to OC and other solid tumors, such asTERT. RNA abundance of the twenty prioritized OC TAAs was determined in307 OC patient samples with available mRNA data expression as describedin Example 29 (FIG. 101B). Fifteen of the prioritized OC TAAs wereexpressed by 100% of samples, 16 TAAs were expressed by 98.0% ofsamples, 17 TAAs were expressed by 79.8% of samples, 18 TAAs wereexpressed by 43.3% of samples, 19 TAAs were expressed by 16.6% ofsamples and 20 TAAs were expressed by 3.9% of samples (FIG. 101C). Thepresent Example thus provides two compositions comprising atherapeutically effective amount of three cancer cell lines, wherein thecombination of the cell lines, a unit dose of six cell lines, comprisescells that express at least 15 TAAs associated with a subset of OCcancer subjects intended to receive said composition. Based on theexpression and immunogenicity data presented herein, the cell linesidentified in Table 84 were selected to comprise the present OC vaccine.

TABLE 84 Ovarian vaccine cell lines and histology Cocktail Cell LineName Histology A OVTOKO Ovarian Clear Cell Carcinoma derived frommetastatic site (spleen) A MCAS Ovarian Mucinous Cystadenocarcinoma ATOV-112D Ovarian Endometrioid Adenocarcinoma B TOV-21G Ovarian ClearCell Carcinoma B ES-2 Ovarian Poorly Differentiated Clear CellAdenocarcinoma B DMS 53 Lung Small Cell Carcinoma

Reduction of CD276 Expression

The OVTOKO, MCAS, TOV-112D, TOV-21G, ES-2, and DMS 53 component celllines expressed CD276 and expression was knocked out by electroporationwith ZFN as described in Example 13 and elsewhere herein. Because it wasdesirable to maintain as much tumor heterogeneity as possible, theelectroporated and shRNA modified cells were not cloned by limitingdilution. Instead, the cells were subjected to multiple rounds of cellsorting by FACS as described in Example 13. Expression of CD276 wasdetermined as described in Example 29. Reduction of CD276 expression isdescribed in Table 85. These data show that gene editing of CD276 withZFN resulted in greater than 98.1% CD276-negative cells in all sixvaccine component cell lines.

TABLE 85 Reduction of CD276 expression Parental Cell Modified Cell %Reduction Cell line Line MFI Line MFI CD276 OVTOKO 108,003 705 99.3 MCAS  2,356  44 98.1 TOV-112D   2,969   7 99.8 TOV-21G  13,475   0 ≥99.9ES-2   3,216   0 ≥99.9 DMS 53  11,928  24 99.8 MFI reported with isotypecontrols subtracted

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12 werecompleted as described in Example 29.

shRNA Downregulates TGF-β Secretion

Following CD276 knockout, TGFβ1 and/or TGFβ2 secretion levels werereduced using shRNA and resulting levels determined as described inExample 29. The OVTOKO, MCAS and TOV-112D parental cell lines in OCvaccine-A secreted measurable levels of TGFβ1 and TGFβ2. The TOV-21G andES-2 component cell lines of OC vaccine-B secreted measurable levels ofTGFβ1 and TGFβ2. Reduction of TGFβ2 secretion by the DMS 53 cell line isdescribed in Example 5 and resulting levels determined as describedabove and herein.

The MCAS, TOV-112D, and ES-2 component cell lines were transduced withTGFβ1 shRNA to decrease TGFβ1 secretion concurrently with the transgeneto increase expression of membrane bound CD40L as described in Example29. MCAS, TOV-112D and ES-2 were also transduced with lentiviralparticles encoding TGFβ2 shRNA to decrease the secretion of TGFβ2 andconcurrently increase expression of GM-CSF (SEQ ID NO: 6) as describedin Example 29. These cells are described by the clonal designation DK6.The OVTOKO and TOV-21G cell lines was transduced with TGFβ1 shRNA todecrease TGFβ1 secretion and concurrently increase expression ofmembrane bound CD40L as described in Example 29. These cells, modifiedto reduce TGFβ1 secretion and not TGFβ2 secretion, are described by theclonal designation DK2. DMS 53 was modified with shRNA to reducesecretion of TGFβ2 as described in Example 26. The J82 and DMS 53 cellsmodified to reduce secretion of TGFβ2 and not TGFβ1 are described by theclonal designation DK4.

Modification of TOV-21G with TGFβ1 shRNA initially decreased TGFβ1secretion, but TGFβ1 secretion was increased after further geneticmodification potentially through a compensatory mechanism to maintaincell proliferation and survival. There was a 19% decrease in TGFβ2secretion by the ES-2 cell line resulting from transduction with TGFβ2shRNA. Immunogenicity of the OC vaccine-B component cell lines TOV-21Gand ES-2 was compared with the immunogenicity of unmodified controls infive HLA diverse donors as described in Example 9. HLA-A and HLA-Balleles for Donors 1-3 is described in Table 74. HLA-A and HLA-B allelesfor the other two donors were as follows: Donor 7, A*03:01 B*07:02 andA*25:01 B*18:01; and Donor 8, A*30:02 B*15:10 and A*30:04 B*58:02. Thedata indicated that the TOV-21G OC vaccine B component cell line wasmore immunogenic (4,390±517 SFU) than unmodified TOV-21G (349±121 SFU)(FIG. 103A). The data further indicated that OC vaccine B component cellline ES-2 was significantly more immunogenic (1,505±394 SFU) thanunmodified ES-2 (238±100 SFU) (p=0.016, Mann-Whitney U) (FIG. 103B). Thedata described above indicate the immunological benefit obtained throughmultiple modifications.

Table 86 shows the percent reduction in TGFβ1 and/or TGFβ2 secretion ingenetically modified component cell lines compared to unmodifiedparental cell lines. If TGFβ1 or TGFβ2 secretion was only detected in 1of 16 replicates run in the ELISA assay the value is reported withoutstandard error of the mean. Gene modification resulted in at least 70%reduction of TGFβ1 secretion (excluding TOV-21G). Gene modification ofTGFβ2 resulted at least 19% reduction in secretion of TGFβ2.

TABLE 86 TGF-β Secretion (pg/10⁶ cells/24 hr) in Component Cell LinesCell Line Cocktail Clone TGFβ1 TGFβ2 OVTOKO A Wild type 517 ± 148 124 ±35  OVTOKO A DK2 157 ± 36  NA OVTOKO A Percent reduction  70% NA MCAS AWild type 1,506 ± 203   871 ± 193 MCAS A DK6 161 ± 35  61 ± 37 MCAS APercent reduction  89%  93% TOV-112D A Wild type 490 ± 91  2,397 ± 635  TOV-112D A DK6 * ≤62 * ≤28 TOV-112D A Percent reduction ≥87% ≥99%TOV-21G B Wild type 1,102 ± 150   526 ± 712 TOV-21G B DK2 1,401 ± 370  NA TOV-21G B Percent reduction NA NA ES-2 B Wild type 987 ± 209 272 ±115 ES-2 B DK6 * ≤19 220 ± 26  ES-2 B Percent reduction ≥98%  19% DMS 53B Wild type 106 ± 10  486 ± 35  DMS 53 B DK4 NA 238 ± 40  DMS 53 BPercent reduction NA  51% DK6: TGFβ1/TGFβ2 double knockdown; DK4: TGFβ2single knockdown; DK2: TGFβ1 single knockdown; * = estimated using LLD,not detected; NA = not applicable.

Based on a dose of 5×10⁵ of each component cell line, the total TGFβ1and TGFβ2 secretion by the modified OC vaccine-A and OC vaccine-B andrespective unmodified parental cell lines are shown in Table 87. Thesecretion of TGFβ1 by OC vaccine-A was reduced by 85% pg/dose/24 hr andTGFβ2 by 94% pg/dose/24 hr. The secretion of TGFβ1 by OC vaccine-A wasreduced by 31% pg/dose/24 hr TGFβ2 by OC vaccine-B was reduced by 23%pg/dose/24 hr.

TABLE 87 Total TGF-β Secretion (pg/dose/24 hr) in OC vaccine-A and OCvaccine-B Cocktail Clones TGFβ1 TGFβ2 A Wild type 1,257 1,696 DK2/DK6  190   107 Percent reduction 85% 94% B Wild type 1,098   642DK2/DK4/DK6   763   492 Percent reduction 31% 23%

GM-CSF Secretion

The MCAS, TOV-112D and ES-2 cell lines were transduced with lentiviralparticles containing both TGFβ2 shRNA and the gene to express GM-CSF(SEQ ID NO: 6) as described above. The OVTOKO and TOV-21G cell lineswere transduced with lentiviral particles to only express GM-CSF (SEQ IDNO: 7). DMS 53 was modified to secrete GM-CSF as described in Example 26and elsewhere herein. The results are shown in Table 87 and describedbelow.

Secretion of GM-CSF increased at least 656-fold in all modifiedcomponent cell lines compared to unmodified, parental cell lines. In OCvaccine-A component cell lines, secretion of GM-CSF increased 656-foldby OVTOKO compared to the parental cell line (≤0.003 ng/10⁶ cells/24hr), 13,280-fold by MCAS compared to the parental cell line (≤0.003ng/10⁶ cells/24 hr), and 1,875-fold by TOV-112D compared to the parentalcell line (≤0.014 ng/10⁶ cells/24 hr). In OC vaccine-B component celllines secretion of GM-CSF increased 426,660-fold by TOV-21G compared tothe parental cell line (≤0.003 ng/10⁶ cells/24 hr), 22,047-fold by ES-2compared to the parental cell line (≤0.003 ng/10⁶ cells/24 hr) and49,313-fold by DMS 53 compared to the parental cell line (≤0.003 ng/10⁶cells/24 hr).

TABLE 88 GM-CSF Secretion in Component Cell Lines GM-CSF GM-CSF CellLine (ng/10⁶ cells/24 hr) (ng/dose/24 hr) OVTOKO   2 ± 0.6   1 MCAS 41 ±13  21 TOV-112D 27 ± 8   14 Cocktail A Total    70  36 TOV-21G 1,382 ±302   691 ES-2 64 ± 19  32 DMS 53 158 ± 15   79 Cocktail B Total 1,604802

Based on a dose of 5×10⁵ of each component cell line, the total GM-CSFsecretion for OC vaccine-A was 36 ng per dose per 24 hours. The totalGM-CSF secretion for OC vaccine-B was 802 ng per dose per 24 hours. Thetotal GM-CSF secretion per dose was therefore 838 ng per 24 hours.

Membrane Bound CD40L (CD154) Expression

The component cell lines were transduced with lentiviral particles toexpress membrane bound CD40L as described above. The methods to detectexpression of CD40L by the five OC cell line components are described inExample 29. Modification of DMS 53 to express membrane bound CD40L isdescribed in Example 15. Evaluation of membrane bound CD40L by all sixvaccine component cell lines is described below. The results shown inFIG. 104 and described below demonstrate CD40L membrane expression wassubstantially increased in all six OC vaccine component cell lines.

Expression of membrane bound CD40L increased at least 288-fold in allcomponent cell lines compared to unmodified, parental cell lines. In OCvaccine-A component cell lines, expression of CD40L increased18,046-fold by OVTOKO (13,661 MFI) compared to the parental cell line (0MFI), 1,068-fold by MCAS (18,150 MFI) compared to the parental cell line(17 MFI), and 288-fold by TOV-112D (288 MFI) compared to the parentalcell line (0 MFI). TOV-112D was subsequently sorted to enrichmembrane-bound CD40L expression. After sorting, expression of membranebound CD40L increased 728-fold compared to the parental cell line. TheTOV-112D component cell line with 288-fold increased expression ofmembrane-bound CD40L was used to generate the described herein and isshown in FIG. 104. In OC vaccine-B component cell lines expression ofCD40L increased 18,874-fold by TOV-21G compared to the parental cellline (0 MFI), 2,823-fold by ES-2 (2,823 MFI) compared to the parentalcell line (0 MFI), and 88,261-fold by DMS 53 (88,261 MFI) compared tothe parental cell line (0 MFI).

IL-12 Expression

The component cell lines were transduced with the IL-12 vector asdescribed in Example 17 and resulting IL-12 p70 expression determined asdescribed above and herein. The results are shown in Table 89 anddescribed below.

Secretion of IL-12 increased at least 1,739-fold in all component celllines modified to secrete IL-12 p70 compared to unmodified, parentalcell lines. In OC vaccine-A component cell lines, secretion of IL-12increased 35-fold by OVTOKO compared to the parental cell line (≤0.0014ng/10⁶ cells/24 hr), 11-fold by MCAS compared to the parental cell line(≤0.001 ng/10⁶ cells/24 hr), and 1,739-fold by TOV-112D compared to theparental cell line (≤0.006 ng/10⁶ cells/24 hr). Expression of IL-12 bythe unmodified TOV-112D cell line was determined in a separateexperiment than secretion of IL-12 by the modified cell line. In OCvaccine-B component cell lines expression of IL-12 increased 137-fold byTOV-21G compared to the parental cell line (≤0.001 ng/10⁶ cells/24 hr)and 43-fold by ES-2 compared to the parental cell line (≤0.001 ng/10⁶cells/24 hr). DMS 53 was not modified to secrete IL-12.

TABLE 89 IL-12 Secretion in Component Cell Lines IL-12 IL-12 Cell Line(ng/10⁶ cells/24 hr) (ng/dose/24 hr) OVTOKO 16 ± 3  8 MCAS 31 ± 7 16TOV-112D 10 ± 7  5 Cocktail A Total 57 29 TOV-21G 38 ± 9 19 ES-2 26 ± 513 DMS 53 NA NA Cocktail B Total 64 32

Based on a dose of 5×10⁵ of each component cell line, the total IL-12secretion for OC vaccine-A was 29 ng per dose per 24 hours. The totalIL-12 secretion for OC vaccine-B was 32 ng per dose per 24 hours. Thetotal IL-12 secretion per dose was therefore 61 ng per 24 hours.

Stable Expression of modTERT by the MCAS Cell Line

As described above, the cells in the vaccine components described hereinwere selected to express a wide array of TAAs, including those known tobe important to antitumor immunity. To further enhance the array ofantigens, the MCAS cell line that was modified to reduce the secretionof TGFβ2, reduce the expression of CD276, and to express GM-CSF,membrane bound CD40L and IL-12 was also transduced with lentiviralparticles expressing the modTERT antigen (SEQ ID NO: 35, SEQ ID NO: 36).The expression of modTERT by MCAS was characterized by flow cytometry.Unmodified and antigen modified cells were stained intracellular with0.03 μg/test anti-rabbit IgG anti-TERT (Abcam ab32020) followed by 0.125ug/test AF647-conjugated donkey anti-rabbit IgG1 antibody (BioLegend#406414). Expression of modTERT increased in the modified cell line(1,558,528 MFI) 6.8-fold over that of the unmodified cell line (227,724MFI) (FIG. 102A).

Stable Expression of modFSHR and modMAGEA10 by the TOV-112D Cell Line

The TOV-112D cell line that was modified to reduce the secretion ofTGFβ1 and TGFβ2, reduce the expression of CD276, and to express GM-CSF,membrane bound CD40L, and IL-12 was also transduced with lentiviralparticles expressing the modFSHR and modMAGEA10 antigens (SEQ ID NO: 43,SEQ ID NO: 44). Expression of modFSHR by TOV-112D was determined by flowcytometry. Unmodified and antigen modified cells were stainedintracellular with 0.03 μg/test anti-mouse IgG1 anti-FSHR antibody(Novus Biologicals, NBP2-36489) followed by 0.125 ug/testAF647-conjugated goat anti-mouse IgG1 antibody (Biolegend #405322).Expression of modFSHR increased in the modified cell line (86,796 MFI)6.6-fold over that of the unmodified cell line (13,249 MFI) (FIG. 102B).Expression of modMAGEA10 by TOV-112D was determined by RT-PCR asdescribed in Example 29 and herein. The forward primer was designed toanneal at the 24-50 bp location in the transgene(ATGCATGCCCGAAGAGGACCTGCAGAG (SEQ ID NO: 132)) and reverse primerdesigned to anneal at the 637-659 bp location in the transgene(GCTCTGCACATCGGACAGCAT (SEQ ID NO: 133)) yielding a 634 bp product.Control primers for β-tubulin are described in Example 29. The geneproduct for modMAGEA10 was detected at the expected size (FIG. 102D) andmRNA increased 141,476-fold relative to the parental control.

Stable Expression of modWT1 and modFOLR1 (modFBP) by the TOV-21G CellLine

The TOV-21G cell line that was modified to reduce the secretion ofTGFβ1, reduce the expression of CD276, and to express GM-CSF, membranebound CD40L, and IL-12 was also transduced with lentiviral particlesexpressing the modWT1 and modFOLR1 antigens (SEQ ID NO: 51, SEQ ID NO:52). Expression of modWT1 by TOV-21G was characterized by flowcytometry. Unmodified and antigen modified cells were stainedintracellular with 0.03 μg/test anti-rabbit IgG1 anti-WT1 antibody(Abcam, ab89901) followed by 0.125 ug/test AF647-conjugated donkeyanti-rabbit IgG1 antibody (BioLegend #406414). Expression of modWT1increased in the modified cell line (687,582 MFI) 4.9-fold over that ofthe unmodified cell line (140,770 MFI) (FIG. 102C).

Expression of modFOLR1 by TOV-21G was determined by RT-PCR as describedin Example 29 and herein. The forward primer was designed to anneal atthe 56-76 bp location in the transgene (GAGAAGTGCAGACCAGAATCG (SEQ IDNO: 130)) and reverse primer designed to anneal at the 588-609 bplocation in the transgene (TCTGCTGTAGTTGGACACCTTG (SEQ ID NO: 131))yielding a 554 bp product. Control primers for β-tubulin are describedin Example 29. The gene product for modFOLR1 was detected at theexpected size (FIG. 102E) and mRNA increased 170,855-fold relative tothe parental control.

Stable Expression of modBORIS by the ES-2 Cell Line

The ES-2 cell line that was modified to reduce the secretion of TGFβ1and TGFβ2, reduce the expression of CD276, and to express GM-CSF,membrane bound CD40L and IL-12 was also transduced with lentiviralparticles expressing the modBORIS antigen (SEQ ID NO: 59, SEQ ID NO:60). Expression of modBORIS by ES-2 was determined by RT-PCR asdescribed in Example 29 and herein. The forward primer was designed toanneal at the 1119-1138 bp location in the transgene(TTCCAGTGCTGCCAGTGTAG (SEQ ID NO: 134)) and reverse primer designed toanneal at the 1559-1578 bp location in the transgene(AGCACTTGTTGCAGCTCAGA (SEQ ID NO: 135)) yielding a 460 bp product.Control primers for β-tubulin are described in Example 29. The geneproduct for modBORIS was detected at the expected size (FIG. 102F) andmRNA increased 4,196-fold relative to the parental control.

Immune Responses to TERT in OC Vaccine-A

IFNγ responses to TERT were evaluated in the context of OC vaccine-A asdescribed in Example 29, and herein, in seven HLA diverse donors(n=4/donor). The HLA-A, HLA-B, and HLA-C alleles for each of the sevendonors are shown in Table 90. IFNγ responses were determined by ELISpotas described in Example 29. IFNγ responses to TERT were determined byELISpot using 15-mers peptides overlapping by 11 amino acids spanningthe entire length of the native TERT antigen (JPT, PM-TERT). IFNγresponses to TERT increased with the modified OC vaccine-A (1047±313SFU) compared to the unmodified OC vaccine-A (707±314 SFU) but did notreach statistical significance (n=7) (FIG. 102G).

Immune Responses to FSHR and MAGEA10 in OC Vaccine-A

IFNγ responses to FSHR and MAGEA10 antigens were evaluated in thecontext of OC vaccine-A as described in Example 29, and herein, in sevenHLA diverse donors (n=4/donor). The HLA-A, HLA-B, and HLA-C alleles foreach of the seven donors are shown in Table 90. IFNγ responses weredetermined by ELISpot as described in Example 29. IFNγ responses to FSHRwere determined by ELISpot using 15-mers peptides overlapping by 9 aminoacids spanning the entire length of the native FSHR antigen purchasedfrom Thermo Scientific Custom Peptide Service. FSHR specific IFNγresponses induced by the modified OC vaccine-A (3,379±1,923 SFU) wereincreased compared to the parental, unmodified OC vaccine-A (709±482SFU) but did not reach statistical significance (n=7) (FIG. 102H). IFNγresponses to MAGEA10 were determined by ELISpot using 15-mers peptidesoverlapping by 9 amino acids spanning the entire length of the nativeMAGEA10 antigen purchased from Thermo Scientific Custom Peptide Service.IFNγ responses to MAGEA10 increased with the modified OC vaccine-A(893±495 SFU) compared to the unmodified OC vaccine-A (630±156 SFU) butdid not reach statistical significance (n=7) (FIG. 102I).

Immune Responses to WT1 and FOLR1 (FBP) in OC Vaccine-B

IFNγ responses to the WT1 and FOLR1 were evaluated in the context ofOC-vaccine B as described in Example 29, and herein, in seven HLAdiverse donors (n=4/donor) (Table 90). IFNγ responses against WT1 andFOLR1 (FBP) were determined by ELISpot using 15-mers peptidesoverlapping by 9 amino acids spanning the entire length of the nativeantigen protein purchased from Thermo Scientific Custom Peptide Service.WT1 specific IFNγ responses were increased by OC vaccine-B (516±241 SFU)compared to the unmodified OC vaccine-B (132±74 SFU) (n=7) but did notreach statistical significance (n=7) (FIG. 102K). FOLR1 (FBP) specificIFNγ responses were increased by OC vaccine-B (467±175 SFU) compared tothe unmodified OC vaccine-B (168±65 SFU) but did not reach statisticalsignificance (n=7) (FIG. 102J).

Immune Responses to BORIS in OC Vaccine-B

IFNγ responses to BORIS were evaluated in the context of OC-vaccine B asdescribed in Example 29, and herein, in seven HLA diverse donors(n=4/donor) (Table 90). IFNγ responses against BORIS were determined byELISpot using 15-mers peptides overlapping by 9 amino acids spanning theentire length of the native antigen protein purchased from ThermoScientific Custom Peptide Service. BORIS specific IFNγ responses weresignificantly increased by OC vaccine-B (2,234±1,011 SFU) compared tothe unmodified OC vaccine-B (121±65 SFU) (p=0.011, Mann-Whitney U test)(n=7) (FIG. 102L).

TABLE 90 Healthy Donor MHC-I characteristics Donor # HLA-A HLA-B HLA-C 1*02:01 *24:02 *08:01 *44:02 *05:01 *07:01 2 *02:01 *25:01 *18:01 *27:05*02:02 *12:03 3 *02:01 *33:01 *07:02 *14:02 *07:02 *08:02 4 *02:01*02:01 *15:01 *51:01 *02:02 *03:04 5 *01:01 *30:01 *08:01 *13:02 *06:02*07:01 6 *02:01 *03:01 *07:02 *44:03 *07:02 *16:01 7 *29:02 *31:01*40:01 *55:01 *03:04 *16:01

Cocktails Induce Immune Responses Against Relevant TAAs

The ability of OC vaccine-A and OC vaccine-B to induce IFNγ responsesagainst ten OC antigens was measured by ELISpot. PBMCs from sevenHLA-diverse healthy donors (Table 90) were co-cultured with autologousDCs loaded with OC vaccine-A or OC vaccine-B for 6 days prior tostimulation with TAA-specific specific peptide pools containing knownMHC-I restricted epitopes. Peptides for stimulation of CD14-PBMCs todetect IFNγ responses to TERT, FSHR, MAGEA10, WT1, FOLR1 and BORIS aredescribed above. Additional 15-mer peptides overlapping by 11 amino acidpeptide pools were sourced as follows: MSLN (GeneScript custom library),Survivin (thinkpeptides, 7769_001-011), PRAME (JPT, PM-01P4) and STEAP1(PM-STEAP1).

FIG. 105 demonstrates the OC vaccine is capable of inducing antigenspecific IFNγ responses in seven HLA-diverse donors to ten OC antigensthat are 3.3-fold more robust (24,942±10,138 SFU) compared to theunmodified parental control (7,495±2,317 SFU) (n=7) (FIG. 105A) (Table91). The unit dose of OC vaccine-A and OC vaccine-B elicited IFNγresponses to seven antigens in one donor, nine antigens in two donorsand ten antigens in four donors (FIG. 106). OC vaccine-A and OCvaccine-B independently demonstrated a 2.2-fold and 6.1-fold increase inantigen specific responses compared to parental controls, respectively.Specifically, OC vaccine-A elicited 12,116±5,813 SFU compared to theunmodified controls (5,385±1,892 SFU) (FIG. 105B). For OC vaccine-A, onedonor responded to six antigens, two donors responded to seven antigens,two donors responded to eight antigens, one donor responded to sevenantigens, and two donors responded ten antigens. OC vaccine-B elicited12,826±4,780 SFU compared to parental controls (2,110±529 SFU) (p=0.011,Mann-Whitney U test) (FIG. 105C). For OC vaccine-B, one donor respondedto six antigens, one donor responded to seven antigens, two donorsresponded to eight antigens, and three donors responded to ten antigens.The present Example thus provides two compositions comprising atherapeutically effective amount of three cancer cell lines, a unit doseof six cell lines, wherein said unit dose is capable of eliciting animmune response 3.3-fold greater than the unmodified compositionspecific to at least seven TAAs expressed in OC patient tumors. OCvaccine-A increased IFNγ responses to at least six TAAs 2.2-fold and OCvaccine-B increased IFNγ responses 6.1-fold to at least six TAAs.

TABLE 91 IFNγ Responses to unmodified and modified OC vaccine componentsDonor Unmodified (SFU ± SEM) Modified (SFU ± SEM) (n = 4) OC vaccine-AOC vaccine-B OC Vaccine OC vaccine-A OC vaccine-B OC Vaccine 1 4,459 ±2,295 260 ± 101 4,719 ± 2,970 386 ± 115 998 ± 446 1,383 ± 537   2 5,910± 1,175 2,240 ± 1,648 7,530 ± 1,735 2,188 ± 1,211 4,801 ± 1,430 6,989 ±2,542 3 2,097 ± 1,631 813 ± 369 2,910 ± 1,933 9,273 ± 2,655 5,615 ±1,764 14,888 ± 4,156  4 5,910 ± 1,175 3,331 ± 1,964 9,241 ± 3,012 22,102± 7,899  27,321 49,423 ± 18,471 5 1,962 ± 863   1,414 ± 617   3,376 ±1,398 42,826 ± 2,276  23,162 ± 7,880  65,985 ± 8,801  6^(∧) 1,418 ±636   1,686 ± 683   4,138 ± 1,060 2,433 ± 1,859 14,107 ± 8,825  22,053 ±12,915 7 16,072 ± 4,222  4,333 ± 1,591 20,405 ± 4,797 ± 1,783 1,358 ±826   6,154 +2,592 ^(∧)n = 3 for Donor 6. All others n = 4

Based on the disclosure and data provided herein, a whole cell vaccinefor Ovarian Cancer comprising the six cancer cell lines, sourced fromATCC or JCRB, OVTOKO (JCRB, JCRB1048), MCAS (JCRB, JCRB0240), TOV-112D(ATCC, CRL-11731), TOV-21G (ATCC, CRL-11730), ES-2 (ATCC, CRL-1978) andDMS 53 (ATCC, CRL-2062) is shown in Table 92. The cell lines representfive ovarian cancer cell lines and one small cell lung cancer (SCLC)cell line (DMS 53). The cell lines have been divided into two groupings:vaccine-A and vaccine-B. Vaccine-A is designed to be administeredintradermally in the upper arm and vaccine-B is designed to beadministered intradermally in the thigh. Vaccine A and B togethercomprise a unit dose of cancer vaccine.

TABLE 92 Cell line nomenclature and modifications TGFβ1 TGFβ2 CD276Cocktail Cell Line KD KD KO GM-CSF CD40L IL-12 TAA(s) A OVTOKO X ND X XX X ND A MCAS X X X X X X X A TOV-112D X X X X X X X B TOV-21G ND ND X XX X X B ES-2 X X X X X X X B DMS 53* ND X X X X X ND ND = Not done.*Cell lines identified as CSC-like cells.

Where indicated in the above table, the genes for the immunosuppressivefactors transforming growth factor-beta 1 (TGFβ1) and transforminggrowth factor-beta 2 (TGFβ2) have been knocked down using shRNAtransduction with a lentiviral vector. The gene for CD276 has beenknocked out by electroporation using zinc-finger nuclease (ZFN) orknocked down using shRNA transduction with a lentiviral vector. Thegenes for granulocyte macrophage—colony stimulating factor (GM-CSF),IL-12, CD40L, modTERT (MCAS), modFSHR (TOV-112D), modMAGEA10 (TOV-112D),modWT1 (TOV-21G), modFOLR1 (modFBP) (TOV-21G) and modBORIS (ES-2) havebeen added by lentiviral vector transduction.

Provided herein are two compositions comprising a therapeuticallyeffective amount of three cancer cell lines, a unit dose of six cancercell lines, modified to reduce the expression of at least oneimmunosuppressive factor and to express at least two immunostimulatoryfactors. One composition, OC vaccine-A, was modified to increase theexpression of three TAAs modhTERT, modFSHR and modMAGEA10. The secondcomposition, OC vaccine-B, was modified to expresses three TAAs, modWT1,modFOLR1 (modFBP) and modBORIS. The unit dose of six cancer cell linesexpresses at least at least 15 TAAs associated with a cancer of a subsetof ovarian cancer subjects intended to receive said composition andinduces IFNγ responses 2.2-fold greater than the unmodified compositioncomponents.

Example 34: Preparation of Squamous Cell Head and Neck Cancer (SCCHN)Cancer Vaccine

This Example demonstrates that reduction of TGFβ1, TGFβ2, and CD276expression with concurrent overexpression of GM-CSF, CD40L, and IL-12 ina vaccine composition of two cocktails, each cocktail composed of threecell lines for a total of 6 cell lines, significantly increased themagnitude of cellular immune responses to at least 10 HN-associatedantigens in an HLA-diverse population. As described herein, the firstcocktail, HN vaccine-A, is composed of cell line HSC-4 that was alsomodified to express modPSMA, cell line HO-1-N-1 that was also modifiedto express modPRAME and modTBXT, and cell line DETROIT 562. The secondcocktail, HN vaccine-B, is composed of cell line KON that was alsomodified to express HPV16 and HPV18 E6/E7, cell line OSC-20, and cellline DMS 53. The six component cell lines collectively express at leasttwenty non-viral antigens, and at least twenty-four, that can provide ananti-HN tumor response.

Identification of HN Vaccine Components

Initial cell line selection criteria identified thirty-five vaccinecomponent cell lines for potential inclusion in the HN vaccine.Additional selection criteria described herein were applied to narrowthe thirty-five cell lines to six cell lines for further evaluation inimmunogenicity assays. These criteria included: endogenous HN associatedantigen expression, lack of expression of additional immunosuppressivefactors, such as IL-10 or IDO1, expression of associated CSC-likemarkers CD44, cMET, ABCG2, LRG5, ALDH1, and BMI-1, ethnicity and age ofthe patient from which the cell line was derived, primary site and stageof the HN cancer, and site from which the cell line was derived (primaryor metastatic).

CSCs play a critical role in the metastasis, treatment resistance, andrelapse of head and neck cancer (Table 2). Expression of TAAs andCSC-like markers by candidate component cell lines was determined by RNAexpression data sourced from the Broad Institute Cancer Cell LineEncyclopedia (CCLE) and from the European Molecular BiologyLaboratory-European Bioinformatics Institute (EMBL-EBI) (OSC-20,HO-1-N-1 and KON). The HGNC gene symbol was included in the CCLE searchand mRNA expression was downloaded for each TAA. Expression of a TAA orCSC-like marker by a cell line was considered positive if the RNA-seqvalue was greater than one (CCLE, FPKM) or zero (EMBL-EBI, TPM).Selection criteria identified six candidate HN vaccine components forfurther evaluation: DETROIT 562, SCC-9, HSC-4, OSC-20, HO-1-N-1 and KON.The six candidate component cell lines expressed nine to seventeen TAAs(FIG. 107A) and four to six CSC-like markers (FIG. 107B). As describedherein, the CSC-like cell line DMS 53 is included as one of the sixvaccine cell lines and expressed fifteen HN TAAs and three HN CSC-likemarkers.

Immunogenicity of the six unmodified HN vaccine component candidates wasevaluated by IFNγ ELISpot as described in Example 9 using three HLAdiverse healthy donors (n=4 per donor). HLA-A and HLA-B alleles for thethree donors were as follows: Donor 1, A*01:01 B*08:01 and A*02:01B*15:01; Donor 2, A*03:01 B*15:01 and A*24:02 B*07:02; Donor 3, A*01:01B*07:02 and A*30:01 B*12:02. KON (1,645±215 SFU) and HSC-4 (1,124±394SFU) were more immunogenic than DETROIT 562 (372±132 SFU), SCC-9 (0±0SFU), OSC-20 (985±265 SFU), and HO-1-N-1 (486±137 SFU) (FIG. 109A).SCC-9 was poorly immunogenic and excluded from further analysis. HSC-4and KON were selected to be included in vaccine cocktail A and vaccinecocktail B, respectively, as described further herein.

Immunogenicity of five selected HN cell lines and the CSC-like cell lineDMS 53 was evaluated in two different combinations of three componentcell lines (FIG. 109C). IFNγ responses were determined against the threecomponent cell lines within the two potential vaccine cocktails by IFNγELISpot as described in Example 8 in five HLA diverse healthy donors(n=4 per donor) (Table 99, Donors 1-3, 5 and 6). IFNγ responses weredetected for both cocktails and to each cell line component in eachcocktail (FIG. 109B). The ability of the individual HN vaccine componentcell lines to induce IFNγ responses against themselves compared to theability of the potential HN vaccine cocktails to induce IFNγ responsesagainst the individual cell lines was also measured by IFNγ ELISpot asdescribed in Examples 8 and 9. There was a trend towards increased IFNγresponses to each HN cell line included in the vaccine cocktails, withthe exception of HSC-4, compared to responses to the cell line alone(FIG. 109D).

The cells in the vaccine described herein were selected to express awide array of TAAs, including those known to be important specificallyfor HN antitumor responses, such as NUF2 or PSMA, and also TAAs known tobe important for targets for HN and other solid tumors, such as TERT.Additionally, one of the six cell lines was also modified to expressHPV16 and 18 viral antigens E6 and E7 since about 25-50% of HNCs areHPV-driven and high risk strains HPV16 and HPV18 contribute to themajority (˜85%) of HPV⁺ HNC cases worldwide. Viral oncoproteins E6 andE7 represent good targets for immunotherapy, as they are continuouslyexpressed by tumor cells and are essential to maintain thetransformation status of HPV+ cancer cells. As shown herein, to furtherenhance the array of TAAs and HPV viral antigens, HSC-4 was modified toexpress modPSMA, HO-1-N-1 was modified to express modPRAME and modTBXT,and KON was modified to express HPV16 and HPV18 E6/E7. TBXT was notendogenously expressed in the six component cell lines at >1.0 FPKMor >0 TPM. HPV16 E6/E7 or HPV18 E6/E7 were not expressed by the HNvaccine component cell lines according to product information providedby ATCC or JCRB. Expression data of the HPV 16 or 18 viral antigens wasnot available in CCLE or EMBL. PSMA was endogenously expressed by one ofthe six component cell lines at >1.0 FPKM or >0 TPM. PRAME wasendogenously expressed by two of the six component cell lines at >1.0FPKM or >0 TPM. (FIG. 107A).

Expression of the transduced antigens modPSMA (FIG. 110A) by HSC-4 (SEQID NO: 37; SEQ ID NO: 38), modPRAME (FIG. 110B) and TBXT (FIG. 120C) byHO-1-N-1 (SEQ ID NO: 65; SEQ ID NO: 66) and HPV16 E6/E7 and HPV18 E6/E7(FIG. 110D) (SEQ ID NO: 67; SEQ ID NO: 68) by KON, were detected by flowcytometry or RT-PCR as described in Example 29 and herein. The modPRAMEand modTBXT antigens are encoded in the same lentiviral transfer vectorseparated by a furin cleavage site (SEQ ID NO: 65 and SEQ ID NO: 66).

Because of the need to maintain maximal heterogeneity of antigens andclonal subpopulations the comprise each cell line, the gene modifiedcell lines utilized in the present vaccine have been established usingantibiotic selection and flow cytometry and not through limitingdilution subcloning.

The endogenous mRNA expression of twenty representative HN TAAs in thepresent vaccine are shown in FIG. 107A. SCC-9 is the only cell line inFIG. 107 that is not included in the present vaccine. The presentvaccine, after introduction antigens described above, expresses of allidentified twenty commonly targeted and potentially clinically relevantTAAs capable of inducing a HN antitumor response. Some of these TAAs areknown to be primarily enriched in HN tumors and some can also induce animmune response to HN and other solid tumors. RNA abundance of thetwenty-four prioritized HN TAAs was determined in 515 HN patient sampleswith available mRNA data expression as described in Example 29 (FIG.108A). Fourteen of the prioritized TAAs were expressed by 100% ofsamples, 15 TAAs were expressed by 97.5% of samples, 16 TAAs wereexpressed by 89.5% of samples, 17 TAAs were expressed by 79.8% ofsamples, 18 TAAs were expressed by 61.0% of samples, 19 TAAs wereexpressed by 35.1% of samples and 20 TAAs were expressed by 10.9% ofsamples (FIG. 108B). Provided herein are two compositions comprising atherapeutically effective amount of three cancer cell lines, wherein thecombination of the cell lines, a unit dose of six cell lines, comprisescells that express at least 14 TAAs associated with a subset of HNcancer subjects intended to receive said composition. Based on theexpression and immunogenicity data presented herein, the cell linesidentified in Table 93 were selected to comprise the present HN vaccine.

TABLE 93 Head and neck vaccine cell lines and histology Cell LineCocktail Name Histology A HSC-4 Tongue Squamous Cell Carcinoma derivedfrom metastatic site (cervical lymph node) A HO-1-N-1 Buccal MucosaSquamous Cell Carcinoma A DETROIT Pharynx Squamous Cell Carcinomaderived 562 from metastatic site (pleural effusion) B KON Mouth FloorSquamous Cell Carcinoma derived from metastatic site (cervical lymphnode) B OSC-20 Tongue Squamous Cell Carcinoma derived from metastaticsite (cervical lymph node) B DMS 53 Lung Small Cell Carcinoma

Reduction of CD276 Expression

The HSC-4, HO-1-N-1, DETROIT 562, KON, OSC-2, and DMS 53 component celllines expressed CD276 and expression was knocked out by electroporationwith ZFN as described in Example 13 and elsewhere herein. Because it wasdesirable to maintain as much tumor heterogeneity as possible, theelectroporated and shRNA modified cells were not cloned by limitingdilution. Instead, the cells were subjected to multiple rounds of cellsorting by FACS as described in Example 13. Expression of CD276 wasdetermined as described in Example 29. Reduction of CD276 expression isdescribed in Table 94. These data show that gene editing of CD276 withZFN resulted in greater than 98.9% CD276-negative cells in all sixvaccine component cell lines.

TABLE 94 Reduction of CD276 expression Parental Cell Line Modified Cell% Reduction Cell line MFI Line MFI CD276 HSC-4 21,934 15 99.9 HO-1-N-112,200 139 98.9 DETROIT 562 9,434 79 99.2 KON 14,762 6 ≥99.9 OSC-208,357 33 99.6 DMS 53 11,928 24 99.8 MFI reported with isotype controlssubtracted

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12 werecompleted as described in Example 29.

shRNA Downregulates TGF-β Secretion

Following CD276 knockout, TGFβ1 and TGFβ2 secretion levels were reducedusing shRNA and resulting levels determined as described in Example 29.The HSC-4, HO-1-N-1 and DETROIT 562 parental cell lines in HN vaccine-Asecreted measurable levels of TGFβ1 and TGFβ2. The KON and OSC-2component cell lines of HN vaccine-B secreted measurable levels of TGFβ1and TGFβ2. OSC-2 secreted low levels of TGFβ1 and was not modified toreduce TGFβ1 secretion. Reduction of TGFβ2 secretion by the DMS 53 cellline is described in Example 26 and resulting levels determined asdescribed above and herein.

The HSC-4, HO-1-N-1, DETROIT 562 and KON component cell lines weretransduced with TGFβ1 shRNA to decrease TGFβ1 secretion and concurrentlyincrease the expression of membrane bound CD40L as described in Example29. The HSC-4, HO-1-N-1, DETROIT 562 and KON were also transduced withlentiviral particles encoding TGFβ2 shRNA to decrease the secretion ofTGFβ2 and concurrently increase expression of GM-CSF (SEQ ID NO: 6) asdescribed in Example 29. These cells are described by the clonaldesignation DK6. Modification of HSC-4 with TGFβ1 shRNA initiallydecreased the secretion of TGFβ1. Subsequent modification of HSC-4 withTGFβ2 shRNA decreased secretion of TGFβ2 but resulted in TGFβ1 secretionlevels similar to the parental cell line (Table 95). TGFβ1 and TGFβ2promote cell proliferation and survival and retaining some TGFβsignaling is likely necessary for proliferation and survival of somecell lines. Immunogenicity of the individual unmodified and modified HNcell vaccine cell line components was evaluated in five HLA diversedonors (Table 99, Donors 1-3, 5 and 6) as described in Example 9. Themodified HSC-4 cell line remained more immunogenic (1,108±628 SFU) thanthe unmodified cell line (400±183 SFU) despite secreting similar TGFβ1levels as the unmodified cell line (FIG. 109E). Increased secretion ofTGFβ1 following reduction of TGFβ2 in the HSC-4 cell line potentiallywas a compensatory survival mechanism. OSC-20 was transduced withlentiviral particles encoding TGFβ2 shRNA to decrease the secretion ofTGFβ2 and concurrently increase expression of GM-CSF (SEQ ID NO: 6) asdescribed in Example 29. OSC-20 was subsequently transduced withlentiviral particles to increase the expression of membrane bound CD40L.DMS 53 was modified with shRNA to reduce secretion of TGFβ2 as describedin Example 26. The OCS-20 and DMS 53 cells modified to reduce secretionof TGFβ2 and not TGFβ1 are described by the clonal designation DK4.

Table 95 shows the percent reduction in TGFβ1 and/or TGFβ2 secretion ingene modified component cell lines compared to unmodified, parental,cell lines. Gene modification resulted in at least 79% reduction ofTGFβ1 secretion. Gene modification of TGFβ2 resulted in at least 51%reduction in secretion of TGFβ2.

TABLE 95 TGF-β Secretion (pg/10⁶ cells/24 hr) in Component Cell LinesCell Line Cocktail Clone TGFβ1 TGFβ2 HSC-4 A Wild type 477 ± 88 252 ±46  HSC-4 A DK6 515 ± 69 * ≤15 HSC-4 A Percent reduction NA 94% HO-1-N-1A Wild type 1,226 ± 183  2,238 ± 488   HO-1-N-1 A DK6 254 ± 60 224 ± 114HO-1-N-1 A Percent reduction 79% 90% DETROIT A Wild type 361 ± 86 1,037± 392   562 DETROIT A DK6 * ≤29 * ≤15 562 DETROIT A Percent reduction≥92%  ≥99%  562 KON B Wild type  863 ± 375 675 ± 243 KON B DK6 * ≤32 268± 148 KON B Percent reduction 96% 60% OSC-2 B Wild type 268 ± 46 1,249 ±383   OSC-2 B DK4 NA 94 ± 31 OSC-2 B Percent reduction NA 92% DMS 53 BWild type 106 ± 10 486 ± 35  DMS 53 B DK4 NA 238 ± 40  DMS 53 B Percentreduction NA 51% DK6: TGFβ1/TGFβ2 double knockdown; DK4: TGFβ2 singleknockdown; DK2: TGFβ1 single knockdown; * = estimated using LLD, notdetected; NA = not applicable

Based on a dose of 5×10⁵ of each component cell line, the total TGFβ1and TGFβ2 secretion by the modified HN vaccine-A and HN vaccine-B andrespective unmodified parental cell lines are shown in Table 96. Thesecretion of TGFβ1 by HN vaccine-A was reduced by 61% and TGFβ2 by 93%pg/dose/24 hr. The secretion of TGFβ1 by HN vaccine-B was reduced by 67%and TGFβ2 by 75% pg/dose/24 hr.

TABLE 96 Total TGF-β Secretion (pg/dose/24 hr) in HN vaccine-A and HNvaccine-B Cocktail Clones TGFβ1 TGFβ2 A Wild type 1,032   1,764 DK6 399127 Percent reduction 61% 93% B Wild type 619 1,205 DK4/DK6 203 300Percent reduction 67% 75%

GM-CSF Secretion

The HSC-4, HO-1-N-1, DETROIT 562 and KON cell lines were transduced withlentiviral particles containing both TGFβ2 shRNA and the gene to expressGM-CSF (SEQ ID NO: 6) as described above. DMS 53 was modified to secreteGM-CSF as described in Example 26 and elsewhere herein. The results areshown in Table 96 and described below.

Secretion of GM-CSF increased at least 9,578-fold in all modifiedcomponent cell lines compared to unmodified, parental cell lines. In HNvaccine-A component cell lines, secretion of GM-CSF increased53,794-fold by HSC-4 compared to the parental cell line (≤0.0042 ng/10⁶cells/24 hr), 13,703-fold by HO-1-N-1 compared to the parental cell line(≤0.0039 ng/10⁶ cells/24 hr), and 13,235-fold by DETROIT 562 compared tothe parental cell line (≤0.0038 ng/10⁶ cells/24 hr). In HN vaccine-Bcomponent cell lines secretion of GM-CSF increased 14,867-fold by KONcompared to the parental cell line (≤0.0047 ng/10⁶ cells/24 hr),9,578-fold by OSC-2 compared to the parental cell line (≤0.0039 ng/10⁶cells/24 hr) and 49,313-fold by DMS 53 compared to the parental cellline (≤0.0032 ng/10⁶ cells/24 hr).

TABLE 97 GM-CSF Secretion in Component Cell Lines GM-CSF GM-CSF CellLine (ng/10⁶ cells/24 hr) (ng/dose/24 hr) HSC-4 226 ± 84  113 HO-1-N-153 ± 11 27 DETROIT 562 50 ± 11 25 Cocktail A Total 329 165 KON 70 ± 2135 OSC-2 37 ± 11 19 DMS 53 158 ± 15  79 Cocktail B Total 265 133

Based on a dose of 5×10⁵ of each component cell line, the total GM-CSFsecretion for HN vaccine-A was 165 ng per dose per 24 hours. The totalGM-CSF secretion for HN vaccine-B was 133 ng per dose per 24 hours. Thetotal GM-CSF secretion per dose was therefore 298 ng per 24 hours.

Membrane Bound CD40L (CD154) Expression

The component cell lines were transduced with lentiviral particles toexpress membrane bound CD40L vector as described above. The methods todetect expression of CD40L by the five HN cell line components aredescribed in Example 29. Modification of DMS 53 to express membranebound CD40L is described in Example 15. Evaluation of membrane boundCD40L by all six vaccine component cell lines is described below. Theresults shown in FIG. 111 and described below demonstrate CD40L membraneexpression was substantially increased in all six HN vaccine componentcell lines.

Expression of membrane bound CD40L increased at least 2,144-fold in allcomponent cell lines compared to unmodified, parental cell lines. In HNvaccine-A component cell lines, expression of CD40L increased18,046-fold by HSC-4 (18,046 MFI) compared to the parental cell line (0MFI), 9,796-fold by HO-1-N-1 (9,796 MFI) compared to the parental cellline (0 MFI), and 18,374-fold by DETROIT 562 (18,374 MFI) compared tothe parental cell line (0 MFI). In HN vaccine-B component cell linesexpression of CD40L increased 15,603-fold by KON compared to theparental cell line (0 MFI), 2,144-fold by OSC-20 (40,738 MFI) comparedto the parental cell line (19 MFI), and 88,261-fold by DMS 53 comparedto the parental cell line (0 MFI).

IL-12 Expression

The component cell lines were transduced with the IL-12 vector asdescribed in Example 17 and resulting IL-12 p70 expression determined asdescribed above and herein. The results are shown in Table 98 anddescribed below.

Secretion of IL-12 increased at least 11,274-fold in all component celllines modified to secrete IL-12 p70 compared to unmodified, parentalcell lines. In HN vaccine-A component cell lines, secretion of IL-12increased 148,017-fold by HSC-4 compared to the parental cell line(≤0.0017 ng/10⁶ cells/24 hr), 33,271-fold by HO-1-N-1 compared to theparental cell line (≤0.0016 ng/10⁶ cells/24 hr), and 21,272-fold byDETROIT 562 compared to the parental cell line (≤0.0015 ng/10⁶ cells/24hr). In HN vaccine-B component cell lines expression of IL-12 increased11,274-fold by KON compared to the parental cell line (≤0.0019 ng/10⁶cells/24 hr) and 22,641-fold by OSC-2 compared to the parental cell line(≤0.0016 ng/10⁶ cells/24 hr). DMS 53 was not modified to secrete IL-12.

TABLE 98 IL-12 Secretion in Component Cell Lines IL-12 IL-12 Cell Line(ng/10⁶ cells/24 hr) (ng/dose/24 hr) HSC-4 249 ± 120 125 HO-1-N-1 52 ±11 26 DETROIT 562 32 ± 6  16 Cocktail A Total 333 167 KON 21 ± 15 11OSC-2 35 ± 12 18 DMS 53 NA NA Cocktail B Total  56 29

Based on a dose of 5×10⁵ of each component cell line, the total IL-12secretion for HN vaccine-A was 167 ng per dose per 24 hours. The totalIL-12 secretion for HN vaccine-B was 29 ng per dose per 24 hours. Thetotal IL-12 secretion per dose was therefore 196 ng per 24 hours.

Stable Expression of modPSMA by the HSC-4 Cell Line

As described above, the cells in the vaccine described herein wereselected to express a wide array of TAAs, including those known to beimportant to antitumor immunity. To further enhance the array ofantigens, the HSC-4 cell line that was modified to reduce the secretionof TGFβ2, reduce the expression of CD276, and to express GM-CSF,membrane bound CD40L and IL-12 was also transduced with lentiviralparticles expressing the modPSMA antigen (SEQ ID NO: 37, SEQ ID NO: 38).

The expression of modPSMA by HSC-4 was characterized by flow cytometry.Unmodified and antigen modified cells were stained intracellular with0.03 μg/test anti-mouse IgG1 anti-PSMA antibody (Abcam, ab268061)followed by 0.125 ug/test AF647-conjugated goat anti-mouse IgG1 antibody(BioLegend #405322). Expression of modPSMA was increased in the modifiedcell line (4,473,981 MFI) 25-fold over that of the parental cell line(174,545 MFI) (FIG. 110A).

Stable Expression of modPRAME and modTBXT by the HO-1-N-1 Cell Line

The HO-1-N-1 cell line that was modified to reduce the secretion ofTGFβ1 and TGFβ2, reduce the expression of CD276, and to express GM-CSF,membrane bound CD40L, and IL-12 was also transduced with lentiviralparticles expressing the modPRAME and modTBXT antigens (SEQ ID NO: 65,SEQ ID NO: 66). Expression of modPRAME by HO-1-N-1 was characterized byflow cytometry. Unmodified and antigen modified cells were stainedintracellular with 0.015 μg/test anti-mouse IgG1 anti-PRAME antibody(Thermo Scientific, MA5-31909) followed by followed by 0.125 ug/testAF647-conjugated goat anti-mouse IgG1 antibody (BioLegend #405322).Expression of modPRAME increased in the modified cell line (290,436 MFI)27-fold over that of the unmodified cell line (10,846 MFI) (FIG. 110B).Expression of modTBXT by HO-1-N-1 was also characterized by flowcytometry. Unmodified and antigen modified cells were stainedintracellular with 0.06 μg/test rabbit anti-TBXT antibody (Abcam,ab209665) followed by 0.125 ug/test AF647-conjugated donkey anti-rabbitIgG1 antibody (BioLegend #406414). Expression of modTBXT increased inthe modified cell line (3,338,324 MFI) 3,338,324-fold over that of theunmodified cell line (0 MFI) (FIG. 110C).

Stable Expression of HPV16 E6/E7 HPV18 E6/E7 by the KON Cell Line

The KON cell line that was modified to reduce the secretion of TGFβ1 andTGFβ2, reduce the expression of CD276, and to express GM-CSF, membranebound CD40L and IL-12 was also transduced with lentiviral particlesexpressing the HPV16 and HPV18 E6 and E7 antigens (SEQ ID NO: 67, SEQ IDNO: 68). Expression of HPV16 and HPV18 E6/E7 by KON was determined byRT-PCR as described in Example 29 and herein. The forward primer todetect HPV16 E6 was designed to anneal at the 33-54 bp location in thetransgene (CCCTCAAGAGAGGCCCAGAAAG (SEQ ID NO: 136)) and reverse primerdesigned to anneal at the 160-182 bp location in the transgene(TACACGATGCACAGGTCCCGGAA (SEQ ID NO: 137)) yielding a 150 bp product.The gene product for HPV16 E6 was detected at the expected size (FIG.110D) and mRNA increased 8,422-fold relative to the parental control.The forward primer to detect HPV16 E7 was designed to anneal at the 1-21bp location in the transgene (CACGGCGATACCCCTACACTG (SEQ ID NO: 138))and reverse primer designed to anneal at the 228-250 bp location in thetransgene (CCATCAGCAGATCTTCCAGGGTT (SEQ ID NO: 139)) yielding a 250 bpproduct. The gene product for HPV16 E7 was detected at the expected size(FIG. 110D) and mRNA increased 7,816-fold relative to the parentalcontrol. The forward primer to detect HPV18 E6 was designed to anneal atthe 59-81 bp location in the transgene (TGAACACCAGCCTGCAGGACATC (SEQ IDNO: 140)) and reverse primer designed to anneal at the 287-312 bplocation in the transgene (GCATCTGATGAGCAGGTTGTACAGGC (SEQ ID NO: 141))yielding a 254 bp product. The gene product for HPV18 E6 was detected atthe expected size (FIG. 110D) and mRNA increased 1,224-fold relative tothe parental control. The forward primer to detect HPV18 E7 was designedto anneal at the 74-97 bp location in the transgene(TGTGCCATGAGCAGCTGTCCGACT (SEQ ID NO: 142)) and reverse primer designedto anneal at the 232-254 bp location in the transgene(AAGGCTCTCAGGTCGTCGGCAGA (SEQ ID NO: 143)) yielding a 181 bp product.The gene product for HPV18 E7 was detected at the expected size (FIG.110D) and mRNA increased 1,684-fold relative to the parental control.Control primers for β-tubulin are described in Example 29.

Immune Responses to PSMA in HN Vaccine-A

IFNγ responses to PSMA were evaluated in the context of HN vaccine-A asdescribed in Example 32, and herein, in six HLA diverse donors(n=4/donor). The HLA-A, HLA-B, and HLA-C alleles for each of the sevendonors are shown in Table 99. IFNγ responses were determined by ELISpotas described in Example 29. PSMA specific IFNγ responses were increasedwith the modified HN vaccine-A (1,433±479 SFU) compared to the parentalunmodified HN vaccine-A (637±369 SFU (FIG. 110E).

Immune Responses to PRAME and TBXT in HN Vaccine-A

IFNγ responses to PRAME and FOLR1 were evaluated in the context ofHN-vaccine A as described in Example 29, and herein, in six HLA diversedonors (n=4/donor) (Table 99). IFNγ responses against modPRAME weredetermined by ELISpot using 15-mer peptides overlapping by 11 aminoacids spanning the entire length of the native antigen protein PRAME(JPT, PM-01P4). modPRAME specific IFNγ responses were increased by HNvaccine-A (687±333 SFU) compared to the unmodified HN vaccine-A (375±314SFU) (FIG. 110F). IFNγ responses to TBXT were determined by ELISpotusing 15-mers peptides overlapping by 11 amino acids (JPT, PM-BRAC)spanning the entire length of the native TBXT antigen. modTBXT specificIFNγ responses were increased by HN vaccine-A (1,071±455 SFU) comparedto the unmodified HN vaccine-A (559±289 SFU) (FIG. 110G).

Immune Responses to HPV16 and HPV18 E6/E7 in HN Vaccine-B

IFNγ responses to the HPV16 and HPV18 E6/E7 antigens introduced into theKON cell line was evaluated in the context of HN-vaccine B as describedin Example 29, and herein, in six HLA diverse donors (n=4/donor) (Table99). Healthy donors from which the immune cells are derived to completethese studies are not screened for HPV16 and HPV18 and responses againstthe HPV16 E6/E7 and HPV18 E6/E7 antigens could be a boosted memoryresponse, and not primed de novo, if the donor was HPV16 or HPV18positive.

IFNγ responses to the HPV16 and HPV18 E6/E7 antigens were determined byELISpot using 15-mers peptides overlapping by 9 amino acids spanning theentire length of the HPV16 and HPV18 E6/E7 antigens purchased fromThermo Scientific Custom Peptide Service. The average IFNγ response toHPV16 E6/E7 was similar with the modified HN vaccine-B (1,974±537 SFU)compared to the unmodified HN vaccine-B (1,845±878 SFU) (FIG. 110H).HPV16 E6/E7 responses were decreased in two of six donors (Donor 2 andDonor 6) primed with HN vaccine-B compared to unmodified HN vaccine-B(FIG. 110I). HPV16 E6/E7 responses were increased with HN vaccine-B inthe other four Donors. It is possible that Donor 2 and Donor 6 wereHPV16 positive and continuous stimulation in the context of the in vitroco-culture assay with HPV16 E6/E7 expressed by HN vaccine-B induced Tcell exhaustion thereby decreasing IFNγ production when stimulated withpeptides in the ELISpot assay. The HN vaccine should not induce T cellexhaustion in HPV16 or HPV18 positive patients because of thedifferences in the mechanism of inducing an immune response in vitro andin vivo. The average IFNγ response to HPV18 E6/E7 was increased bymodified HN vaccine-B (2,195±757 SFU) compared to the unmodified HNvaccine-B (822±342 SFU) (FIG. 110J). HPV18 E6/E7 responses weredecreased in Donor 6 when primed with HN vaccine-B compared tounmodified HN vaccine-B but increased in the other five Donors (FIG.110K).

TABLE 99 Healthy Donor MHC-I characteristics Donor # HLA-A HLA-B HLA-C 1*02:01 *02:01 *15:01 *51:01 *02:02 *03:04 2 *02:01 *03:01 *07:02 *49:01*07:01 *07:02 3 *03:01 *32:01 *07:02 *15:17 *07:01 *07:02 4 *01:01*30:01 *08:01 *13:02 *06:02 *07:02 5 *02:01 *30:02 *14:02 *13:02 *08:02*18:02 6 *30:02 *30:04 *15:10 *58:02 *03:04 *06:02

Cocktails Induce Immune Responses Against Relevant TAAs

The ability of HN vaccine-A and HN vaccine-B to induce IFNγ productionagainst ten HN antigens was measured by ELISpot. PBMCs from sixHLA-diverse healthy donors (Table 99) were co-cultured with autologousDCs loaded with HN vaccine-A or HN vaccine-B for 6 days prior tostimulation with TAA-specific specific peptide pools containing knownMHC-I restricted epitopes. Peptides for stimulation of CD14-PBMCs todetect IFNγ responses to PSMA, PRAME, TBXT, HPV16 E6/E7 and HPV18 E6/E7are described above. Additional 15-mer peptides overlapping by 11 aminoacid peptide pools were sourced as follows: Survivin (thinkpeptides,7769_001-011), MUC1 (JPT, PM-MUC1), and STEAP1 (PM-STEAP1).

FIG. 112 demonstrates the HN vaccine is capable of inducing antigenspecific IFNγ responses in six HLA-diverse donors to ten HN antigensthat are 1.6-fold more robust (18,901±3,963 SFU) compared to theunmodified parental control (11,537±5,281 SFU) (FIG. 109A) (Table 100).The HN vaccine also increased IFNγ responses to non-viral antigens1.6-fold (10,331±2,342 SFU) compared to unmodified parental control(6,568±3,112 SFU) (FIG. 112D). The unit dose of HN vaccine-A and HNvaccine-B elicited IFNγ responses to nine antigens in one donor and tenantigens in five donors (FIGS. 113A and 113B, upper panel). The unitdose of HN vaccine-A and HN vaccine-B elicited IFNγ responses to fivenon-viral antigens in one donor and six non-viral antigens in fivedonors (FIGS. 113A and 113B, lower panel) (Table 101). HN vaccine-A andHN vaccine-B independently demonstrated a 1.7-fold and 1.6-fold increasein antigen specific responses compared to parental controls,respectively, for all antigens.

HN vaccine-A and HN vaccine-B independently demonstrated a 1.5-fold and1.6-fold increase in non-viral antigen specific responses compared toparental controls, respectively. Specifically, HN vaccine-A elicited9,843±2,539 SFU compared to the unmodified controls (5,848±3,222 SFU)for all antigens and (FIG. 112B) (Table 100) and 5,441±1,694 SFU tonon-viral antigens compared to the unmodified controls (3,547±1,990 SFU)(FIG. 112E) (Table 101). For HN vaccine-A, one donor responded to sevenantigens, two donors responded to nine antigens, and three donorsresponded to ten antigens. HN vaccine-A elicited IFNγ responses to fournon-viral antigens in one donor, five non-viral antigens in two donorsand six non-viral antigens in three donors. HN vaccine-B elicited9,058±1,715 SFU compared to the unmodified controls (5,688±2,472 SFU)for all antigens and (FIG. 112C) (Table 100) and 4,890±932 SFU tonon-viral antigens compared to the unmodified controls (3,022±1,333 SFU)(FIG. 112F) (Table 101). For HN vaccine-B, one donor responded to sixantigens, one donor responded to seven antigens, and two donorsresponded to nine antigens and two donors responded to ten antigens. HNvaccine-A elicited IFNγ responses to three non-viral antigens in onedonor, four non-viral antigens in one donor, five non-viral antigens intwo donors and six non-viral antigens in two donors.

Described above are two compositions comprising a therapeuticallyeffective amount of three cancer cell lines, a unit dose of six celllines, wherein said unit dose is capable of eliciting an immune response1.6-fold greater than the unmodified composition specific to at leastnine TAAs expressed in HN patient tumors. HN vaccine-A increased IFNγresponses to at least seven TAAs 1.7-fold and HN vaccine-B increasedIFNγ responses 1.6-fold to at least six TAAs.

TABLE 100 IFNγ Responses to unmodified and modified HN vaccinecomponents Donor Unmodified (SFU ± SEM) Modified (SFU ± SEM) (n = 4) HNvaccine-A HN vaccine-B HN Vaccine HN vaccine-A HN vaccine-B HN Vaccine 1542 ± 306 2,149 ± 1,421 2,691 ± 1,718 4,209 ± 1,876 10,106 ± 2,386 14,314 ± 1,483  2 20,690 ± 3,007  15,873 ± 4,506  36,563 ± 6,481  17,240± 5,541  14,265 ± 3,225  31,505 ± 3,770  3 3,500 ± 2,201 2,663 ± 450  6,133 ± 2,484 6,055 ± 2,562 8,905 ± 2,398 14,960 ± 4,113  4 8,620 ±2,267 2,158 ± 1,092 10,778 ± 2,642  15,348 ± 5,682  10,780 ± 2,484 26,128 ± 7,506  5 520 ± 263 903 ± 572 1,423 ± 802   2,800 ± 1,336 1,513± 725   4,313 ± 1,640 6 1,218 ± 652   10,415 ± 3,103  11,633 ± 3,700 13,405 ± 2,355  8,783 ± 3,081 22,188 ± 2,851 

TABLE 101 IFNγ Responses to non-viral antigens by unmodified andmodified HN vaccine components Donor Unmodified (SFU ± SEM) Modified(SFU ± SEM) (n = 4) HN vaccine-A HN vaccine-B HN Vaccine HN vaccine-A HNvaccine-B HN Vaccine 1 405 ± 313 1,408 ± 1,127 1,294 ± 737   1,003 ±420   5,078 ± 833   3,709 ± 1,241 2 12,757 ± 2,435  8,840 ± 2,337 16,102± 3,210  10,984 ± 3,964  4,863 ± 1,434 12,679 ± 4,886  3 2,223 ± 1,2781,583 ± 294   2,815 ± 1,076 2,700 ± 1,782 4,568 ± 1,385 4,813 ± 1,368 45,098 ± 1,131 683 ± 383 5,013 ± 1,069 8,513 ± 2,941 6,770 ± 1,525 9,655± 2,574 5 168 ± 89  665 ± 344 958 ± 516 1,745 ± 692   795 ± 541 2,493 ±1,137 6 630 ± 514 4,953 ± 1,337 6,008 ± 1,624 7,700 ± 692   7,265 ±1,702 13,590 ± 1,823 

Based on the disclosure and data provided herein, a whole cell vaccinefor Head and Neck Cancer comprising the six cancer cell lines, sourcedfrom ATCC or JCRB, HSC-4 (JCRB, JCRB0624), HO-1-N-1 (JCRB, JCRB0831),DETROIT 562 (ATCC, CCL-138), KON (JCRB, JCRB0194), OSC-20 (JCRB,JCRB0197) and DMS 53 (ATCC, CRL-2062) is shown in Table 101A. The celllines represent five head and neck cancer cell lines and one small celllung cancer (SCLC) cell line (DMS 53 ATCC CRL-2062). The cell lines havebeen divided into two groupings: vaccine-A and vaccine-B. Vaccine-A isdesigned to be administered intradermally in the upper arm and vaccine-Bis designed to be administered intradermally in the thigh. Vaccine A andB together comprise a unit dose of cancer vaccine.

TABLE 101A Cell line nomenclature and modifications TGFβ1 TGFβ2 CD276Cocktail Cell Line KD KD KO GM-CSF CD40L IL-12 TAA(s) A HSC-4* X X X X XX X A HO-1-N-1 X X X X X X X A DETROIT X X X X X X ND 562* B KON X X X XX X X B OSC-20 ND X X X X X ND B DMS 53* ND X X X X X ND ND = Not done.*Cell lines identified as CSC-like cells.

Where indicated in the above table, the genes for the immunosuppressivefactors transforming growth factor-beta 1 (TGFβ1) and transforminggrowth factor-beta 2 (TGFβ2) have been knocked down using shRNAtransduction with a lentiviral vector. The gene for CD276 has beenknocked out by electroporation using zinc-finger nuclease (ZFN) orknocked down using shRNA transduction with a lentiviral vector. Thegenes for granulocyte macrophage—colony stimulating factor (GM-CSF),IL-12, CD40L, modPSMA (HSC-4), modPRAME (HO-1-N-1), modTBXT (HO-1-N-1),HPV16 E6 and E7 (KON) and HPV18 E6 and E7 (KON) were added by lentiviralvector transduction.

Provided herein are two compositions comprising a therapeuticallyeffective amount of three cancer cell lines, a unit dose of six cancercell lines, modified to reduce the expression of at least twoimmunosuppressive factors and to express at least two immunostimulatoryfactors. One composition, HN vaccine-A, was modified to increase theexpression of three TAAs, modPSMA, modPRAME and modTBXT. The secondcomposition, HN vaccine-B, was modified to expresses four viral tumorassociated antigens, HPV16 E6 and E7 and HPV18 E6 and E7. The unit doseof six cancer cell lines expresses at least at least 14 non-viral TAAsassociated with a cancer of a subset of head and neck cancer subjectsintended to receive said composition and induces IFNγ responses 1.6-foldgreater than the unmodified composition components.

Example 35: Preparation of Gastric Cancer Vaccine

This Example demonstrates that reduction of TGFβ1, TGFβ2, and CD276expression with concurrent overexpression of GM-CSF, CD40L, and IL-12 ina vaccine composition of two cocktails, each cocktail composed of threecell lines for a total of 6 cell lines, significantly increased themagnitude of cellular immune responses to at least 10 GCA-associatedantigens in an HLA-diverse population. As described herein, the firstcocktail, GCA vaccine-A, is composed of cell line MKN-1 that was alsomodified to express modPSMA and modLYK6, cell line MKN-45, and cell lineMKN-74. The second cocktail, GCA vaccine-B, is composed of cell lineOCUM-1, cell line Fu97 that was also modified to express modWT1 andmodCLDN18 (Claudin 18), and cell line DMS 53. The six component celllines collectively express at least twenty antigens that can provide ananti-GCA tumor response.

Identification of GCA Vaccine Components

Initial cell line selection criteria identified thirty-six vaccinecomponent cell lines for potential inclusion in the GCA vaccine.Additional selection criteria described herein were applied to narrowthe thirty-six cell lines to seven cell lines for further evaluation inimmunogenicity assays. These criteria included: endogenous GCAassociated antigen expression, lack of expression of additionalimmunosuppressive factors, such as IL-10 or IDO1, expression ofGCA-associated CSC-like markers ABCB1, ABCG2, ALDH1A, CD133, CD164,FUT4, LGR5, CD44, MUC1 and DLL4, ethnicity and age of the patient fromwhich the cell line was derived, cancer stage and site from which thecell line was derived, and histological subtype.

CSCs play a critical role in the metastasis, treatment resistance, andrelapse of gastric cancer (Table 2). Expression of TAAs and GCA specificCSC-like markers by candidate component cell lines was determined by RNAexpression data sourced from the Broad Institute Cancer Cell LineEncyclopedia (CCLE). The HGNC gene symbol was included in the CCLEsearch and mRNA expression was downloaded for each TAA. Expression of aTAA or CSC-like marker by a cell line was considered positive if theRNA-seq value was greater than one. Selection criteria identified sevencandidate GCA vaccine components for further evaluation: RERF-GC-1B,MKN-74, MKN-45, OCUM-1, MKN-1, Fu97 and NCI-N87. The seven candidatecomponent cell lines expressed ten to fourteen TAAs (FIG. 114A) and twoto six CSC markers (FIG. 114B). As described herein, the CSC-like cellline DMS 53 is included as one of the six vaccine cell lines andexpressed fourteen GCA TAAs and seven GCA CSC-like markers.

Immunogenicity of the seven unmodified GCA vaccine component candidateswas evaluated by IFNγ ELISpot as described in Example 9 using three HLAdiverse healthy donors (n=4 per donor). HLA-A and HLA-B alleles forDonors were as follows: Donor 1, A*01:01 B*08:01 and A*02:01 B*15:01;Donor 2, A*01:01 B*08:01 and A*02:01 B*57:03; and Donor 3, A*02:01B*40:01 and A*30:01 B*57:01. MKN-1 (5,417±152 SFU) and OCUM-1 (1,123±258SFU) were more immunogenic than RERF-GC-1B (120±56 SFU), MKN-74 (241±107SFU), MKN-45 (0±0 SFU), Fu97 (578±209 SFU) and NCI-N87 (0±0 SFU) (FIG.115A).

Immunogenicity of MKN-1 and OCUM-1 was evaluated in eight differentcombinations of three component cell lines, four combinations containedMKN-1 and four combinations contained OCUM-1 (FIG. 115C). IFNγ responseswere determined against the three component cell lines within the eightpotential vaccine cocktails by IFNγ ELISpot as described in Example 8using the three healthy donors (n=4/donor). HLA-A and HLA-B alleles forthe donors were as follows: Donor 1, A*02:06 B*15:01 and A*34:02B*51:01; Donor 2, A*03:01 B*07:02 and A*24:02 B*15:09; and Donor 3,A*02:01 B*40:01 and A*30:01 B*57:01. IFNγ responses were detected forall eight cocktails and to each cell line component in each cocktail.Responses to the individual cocktail component cell lines were notablyincreased for most cell lines compared to IFNγ responses detected forsingle cell line components (FIG. 115B). In all eight combinationsevaluated, MKN-1 remained the most immunogenic. MKN-1 was selected to beincluded in vaccine cocktail A and OCUM-1 was selected to be included invaccine cocktail B as described above and further herein.

The cells in the vaccine described herein were selected to express awide array of TAAs, including those known to be important specificallyfor GCA antitumor responses, such as LY6K or MUC1, and also TAAs knownto be important for targets for GCA and other solid tumors, such TERT.As shown herein, to further enhance the array of TAAs, MKN-1 wasmodified to express modPSMA and modLY6K, and Fu97 was modified toexpress modWT1 and modCLDN18. PSMA, CLDN18 and WT1 were endogenouslyexpressed by one of the six component cell lines and LY6K wasendogenously expressed by two of the six component cell lines at >1.0FPKM (FIG. 116A).

Expression of the transduced antigens modPSMA (FIG. 117A) and modLY6K(FIG. 117B) by MKN-1 (SEQ ID NO: 57; SEQ ID NO: 58), and modWT1 (FIG.114C) and modCLDN18 (FIG. 114D) (SEQ ID NO: 55; SEQ ID NO: 56) by Fu97,were detected by flow cytometry as described in Example 29 and herein.The modPSMA and modLY6K antigens are encoded in the same lentiviraltransfer vector separated by a furin cleavage site. The modWT1 andmodCLDN18 are encoded in the same lentiviral transfer vector separatedby a furin cleavage site.

Because of the need to maintain maximal heterogeneity of antigens andclonal subpopulations the comprise each cell line, the gene modifiedcell lines utilized in the present vaccine have been established usingantibiotic selection and flow cytometry and not through limitingdilution subcloning.

The endogenous mRNA expression of twenty representative GCA TAAs in thepresent vaccine are shown in FIG. 116A. The present vaccine, afterintroduction antigens described above, expresses of all identifiedtwenty commonly targeted and potentially clinically relevant TAAscapable of inducing a GCA antitumor response. Some of these TAAs areknown to be primarily enriched in GCA tumors and some can also induce animmune response to GCA and other solid tumors. RNA abundance of thetwenty prioritized GCA TAAs was determined in 371 GCA patient sampleswith available mRNA data expression as described in Example 29 (FIG.116B). Eleven of the prioritized GCA TAAs were expressed by 100% ofsamples, 12 TAAs were expressed by 99.5% of samples, 13 TAAs wereexpressed by 98.9% of samples, 14 TAAs were expressed by 94.9% ofsamples, 15 TAAs were expressed by 83.0% of samples, 16 TAAs wereexpressed by 67.1% of samples, 17 TAAs were expressed by 43.4% ofsamples, 18 TAAs were expressed by 24.5% of samples, 19 TAAs wereexpressed by 8.6% of samples and 20 TAAs were expressed by 0.3% ofsamples (FIG. 116C). Provided herein are two compositions comprising atherapeutically effective amount of three cancer cell lines, wherein thecombination of the cell lines, a unit dose of six cell lines, comprisescells that express at least 11 TAAs associated with a subset of GCAcancer subjects intended to receive said composition. Based on theexpression and immunogenicity data presented herein, the cell linesidentified in Table 102 were selected to comprise the present GCAvaccine.

TABLE 102 Gastric vaccine cell lines and histology Cell Line CocktailName Histology A MKN-1 Gastric Adenocarcinoma; derived from metastaticsite (lymph node) A MKN-45 Gastric Adenocarcinoma; derived frommetastatic site (liver) A MKN-74 Gastric Tubular Adenocarcinoma B OCUM-1Signet Ring Cell Gastric Adenocarcinoma; derived from metastatic site(pleural effusion) B Fu97 Gastric Adenocarcinoma; derived frommetastatic site (lymph node) B DMS 53 Lung Small Cell Carcinoma

Reduction of CD276 Expression

The MKN-1, MKN-45, MKN-74, OCUM-1, FU97, and DMS 53 component cell linesexpressed CD276 and expression was knocked out by electroporation withZFN as described in Example 13 and elsewhere herein. Because it wasdesirable to maintain as much tumor heterogeneity as possible, theelectroporated and shRNA modified cells were not cloned by limitingdilution. Instead, the cells were subjected to multiple rounds of cellsorting by FACS as described in Example 13. Expression of CD276 wasdetermined as described in Example 29. Reduction of CD276 expression isdescribed in Table 103. These data show that gene editing of CD276 withZFN resulted in greater than 83.3% CD276-negative cells in all sixvaccine component cell lines.

TABLE 103 Reduction of CD276 expression Parental Cell Line Modified Cell% Reduction Cell line MFI Line MFI CD276 MKN-1 35,503 6 ≥99.9 MKN-458,479 11 99.9 MKN-74 11,335 3 ≥99.9 OCUM-1 13,474 2,244 83.3 FU97178,603 394 99.8 DMS 53 11,928 24 99.8 MFI reported with isotypecontrols subtracted

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12 CytokineSecretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12 were completed asdescribed in Example 29.

shRNA Downregulates TGF-β Secretion

Following CD276 knockout, TGFβ1 and TGFβ2 secretion levels were reducedusing shRNA and resulting levels determined as described in Example 26.The MKN-1, MKN-45, and MKN-74 cell lines in GCA vaccine-A secretedmeasurable levels of TGFβ1. MKN-1 also secreted measurable levels ofTGFβ2. The Fu97 and DMS 53 component cell lines of GCA vaccine-Bsecreted measurable levels of TGFβ1. DMS 53 also secreted measurablelevels of TGFβ2. OCUM-1 did not secrete measurable levels of TGFβ1 orTGFβ2. Reduction of TGFβ2 secretion by the DMS 53 cell line is describedin Example 26 and resulting levels determined as described above andherein.

The MKN-1 component cell lines were transduced with TGFβ1 shRNA todecrease TGFβ1 secretion concurrently with the transgene to increaseexpression of membrane bound CD40L as described in Example 29. MKN-1 wasalso transduced with lentiviral particles encoding TGFβ2 shRNA todecrease the secretion of TGFβ2 and concurrently increase expression ofGM-CSF (SEQ ID NO: 6) as described in Example 29. These cells aredescribed by the clonal designation DK6. The MKN-45, MKN-74 and Fu97cell lines were transduced with TGFβ1 shRNA to decrease TGFβ1 secretionand concurrently increase expression of membrane bound CD40L asdescribed in Example 29. These cells, modified to reduce TGFβ1 secretionand not TGFβ2 secretion, are described by the clonal designation DK2.DMS 53 was modified with shRNA to reduce secretion of TGFβ2 as describedin Example 26. Modification of DMS 53 cells to reduce secretion of TGFβ2and not TGFβ1 are described by the clonal designation DK4. OCUM-1 wasnot modified to reduce TGFβ1 or TGFβ2 secretion because the parentalline did not secrete detective levels of TGFβ1 or TGFβ2.

Table 104 shows the percent reduction in TGFβ1 and/or TGFβ2 secretion ingene modified component cell lines compared to unmodified parental, celllines. Gene modification resulted in at least 72% reduction of TGFβ1secretion. Gene modification of TGFβ2 resulted in at least 51% reductionin secretion of TGFβ2.

TABLE 104 TGF-β Secretion (pg/10⁶ cells/24 hr) in Component Cell LinesCell Line Cocktail Clone TGFβ1 TGFβ2 MKN-1 A Wild type 2,539 ± 670 1,634 ± 670  MKN-1 A DK6 218 ± 58 * >12 MKN-1 A Percent reduction 91%≥99%  MKN-45 A Wild type  704 ± 101 * >11 MKN-45 A DK2  98 ± 49 NAMKN-45 A Percent reduction 86% NA MKN-74 A Wild type  753 ± 104  * >6MKN-74 A DK2 119 ± 18 NA MKN-74 A Percent reduction 84% NA OCUM-1 B Wildtype * >22 * >10 OCUM-1 B NA NA NA OCUM-1 B Percent reduction NA NA Fu97B Wild type  402 ± 103 * >11 Fu97 B DK2 113 ± 14 NA Fu97 B Percentreduction 72% NA DMS 53 B Wild type 106 ± 10 486 ± 35 DMS 53 B DK4 NA238 ± 40 DMS 53 B Percent reduction NA 51% DK6: TGFβ1/TGFβ2 doubleknockdown; DK4: TGFβ2 single knockdown; DK2: TGFβ1 single knockdown; * =estimated using LLD, not detected; NA = not applicable

Based on a dose of 5×10⁵ of each component cell line, the total TGFβ1and TGFβ2 secretion by the modified GCA vaccine-A and GCA vaccine-B andrespective unmodified parental cell lines are shown in Table 105. Thesecretion of TGFβ1 by GCA vaccine-A was reduced by 89% and TGFβ2 by 98%pg/dose/24 hr. The secretion of TGFβ1 by GCA vaccine-B was reduced by54% and TGFβ2 by 49% pg/dose/24 hr.

TABLE 105 Total TGF-β Secretion (pg/dose/24 hr) in GCA vaccine-A and GCAvaccine-B Cocktail Clones TGFβ1 TGFβ2 A Wild type 1,998   826 DK2/DK6218  15 Percent reduction 89% 98% B Wild type 265 254 DK2/DK4 121 130Percent reduction 54% 49%

GM-CSF Secretion

The MKN-1 cell line was transduced with lentiviral particles containingboth TGFβ2 shRNA and the gene to express GM-CSF (SEQ ID NO: 6) asdescribed above. The MKN-45, MKN-74, OCUM-1 and Fu97 cell lines weretransduced with lentiviral particles to only express GM-CSF (SEQ ID NO:7). DMS 53 was modified to secrete GM-CSF as described in Example 26 andelsewhere herein. The results are shown in Table 106 and describedbelow.

Secretion of GM-CSF increased at least 3,941-fold in all modifiedcomponent cell lines compared to unmodified parental cell lines. In GCAvaccine-A component cell lines, secretion of GM-CSF increased46,419-fold by MKN-1 compared to the parental cell line (≤0.0028 ng/10⁶cells/24 hr), 3,941-fold by MKN-45 compared to the parental cell line(≤0.0051 ng/10⁶ cells/24 hr), and 242,155-fold by MKN-74 compared to theparental cell line (≤0.0027 ng/10⁶ cells/24 hr). In GCA vaccine-Bcomponent cell lines secretion of GM-CSF increased 7,866-fold by OCUM-1compared to the parental cell line (≤0.0043 ng/10⁶ cells/24 hr),193,248-fold by Fu97 compared to the parental cell line (≤0.0046 ng/10⁶cells/24 hr) and 49,313-fold by DMS 53 compared to the parental cellline (≤0.0032 ng/10⁶ cells/24 hr).

TABLE 106 GM-CSF Secretion in Component Cell Lines GM-CSF GM-CSF CellLine (ng/10⁶ cells/24 hr) (ng/dose/24 hr) MKN-1 130 ± 66  65 MKN-45 20 ±8  10 MKN-74 664 ± 374 332 Cocktail A Total 814 407 OCUM-1 34 ± 17 17FU97 893 ± 422 447 DMS 53 158 ± 15  79 Cocktail B Total 1,085   543

Based on a dose of 5×10⁵ of each component cell line, the total GM-CSFsecretion for GCA vaccine-A was 407 ng per dose per 24 hours. The totalGM-CSF secretion for GCA vaccine-B was 543 ng per dose per 24 hours. Thetotal GM-CSF secretion per dose was therefore 950 ng per 24 hours.

Membrane Bound CD40L (CD154) Expression

The component cell lines were transduced with lentiviral particles toexpress membrane bound CD40L vector as described above. The methods todetect expression of CD40L by the five GCA cell line components aredescribed in Example 29. Modification of DMS 53 to express membranebound CD40L is described in Example 15. Evaluation of membrane boundCD40L by all six vaccine component cell lines is described below. Theresults shown in FIG. 118 and described below demonstrate CD40L membraneexpression was substantially increased in all six GCA vaccine componentcell lines.

Expression of membrane bound CD40L increased at least 374-fold in allcomponent cell lines compared to unmodified, parental cell lines. In GCAvaccine-A component cell lines, expression of CD40L increased15,941-fold by MKN-1 (15,941 MFI) compared to the parental cell line (0MFI), 374-fold by MKN-45 (3,397 MFI) compared to the parental cell line(9 MFI), and 4,914-fold by MKN-74 (4,914 MFI) compared to the parentalcell line (0 MFI). In GCA vaccine-B component cell lines expression ofCD40L increased 3,741-fold by OCUM-1 (3,741 MFI) compared to theparental cell line (0 MFI), 1,569-fold by FU97 (26,449 MFI) compared tothe parental cell line (17 MFI), and 88,261-fold by DMS 53 compared tothe parental cell line (0 MFI).

IL-12 Expression

The MKN-1, MKN-45, MKN-74, and Fu97 component cell lines were transducedwith the IL-12 vector as described in Example 17 and resulting IL-12 p70expression determined as described above and herein. The results areshown in Table 107 and described below.

Secretion of IL-12 increased at least 1,715-fold in all component celllines modified to secrete IL-12 p70 compared to unmodified, parentalcell lines. In GCA vaccine-A component cell lines, secretion of IL-12increased 53,185-fold by MKN-1 compared to the parental cell line(≤0.0011 ng/10⁶ cells/24 hr), 1,715-fold by MKN-45 compared to theparental cell line (≤0.0021 ng/10⁶ cells/24 hr), and 56,743-fold byMKN-74 compared to the parental cell line (≤0.0011 ng/10⁶ cells/24 hr).In GCA vaccine-B component cell lines expression of IL-12 increased13,078-fold by FU97 compared to the parental cell line (≤0.0037 ng/10⁶cells/24 hr). OCUM-1 and DMS 53 were not modified to secrete IL-12.

TABLE 107 IL-12 Secretion in Component Cell Lines IL-12 IL-12 Cell Line(ng/10⁶ cells/24 hr) (ng/dose/24 hr) MKN-1 60 ± 25 30 MKN-45 4 ± 2  2MKN-74 62 ± 7  31 Cocktail A Total 126  63 OCUM-1 NA NA FU97 48 ± 11 24DMS 53 NA NA Cocktail B Total 48 24

Based on a dose of 5×10⁵ of each component cell line, the total IL-12secretion for GCA vaccine-A was 63 ng per dose per 24 hours. The totalIL-12 secretion for GCA vaccine-B was 24 ng per dose per 24 hours. Thetotal IL-12 secretion per dose was therefore 87 ng per 24 hours.

Stable Expression of modPSMA and modLY6K by the MKN-1 Cell Line

As described above, the cells in the vaccine described herein wereselected to express a wide array of TAAs, including those known to beimportant to antitumor immunity. To further enhance the array ofantigens, the MKN-1 cell line that was modified to reduce the secretionof TGFβ1 and TGFβ2, reduce the expression of CD276, and to expressGM-CSF, membrane bound CD40L and IL-12 was also transduced withlentiviral particles expressing the modPSMA and modLY6K antigens. RNAexpression data sourced from CCLE suggested that MKN-1 endogenouslyexpressed LYK6 (FIG. 116A) but the LYK6 protein was not detected inunmodified MKN-1 cells by flow cytometry (FIG. 117B). The genes encodingthe modPSMA and modLY6K antigens (SEQ ID NO: 57, SEQ ID NO: 58) arelinked by a furin cleavage site.

The expression of modPSMA by MKN-1 was characterized by flow cytometry.Unmodified and antigen modified cells were stained intracellular with0.03 μg/test anti-mouse IgG1 anti-PSMA antibody (Abcam, ab268061)followed by 0.125 ug/test AF647-conjugated goat anti-mouse IgG1 antibody(BioLegend #405322). Expression of modPSMA was increased in the modifiedcell line (697,744 MFI) 15-fold over that of the parental cell line(46,955 MFI) (FIG. 117A). Expression of modLY6K by MKN-1 was alsocharacterized by flow cytometry. Cells were first stained intracellularwith rabbit IgG anti-LY6K antibody (Abcam, ab246486) (0.03 μg/test)followed by AF647-conjugated donkey anti-rabbit IgG1 antibody (BioLegend#406414) (0.125 μg/test). Expression of modLY6K increased in themodified cell line (2,890,315 MFI) 2,890,315-fold over the unmodifiedcell line (0 MFI) (FIG. 117B).

Stable Expression of modWT1 and modCLDN18 by the Fu97 Cell Line

The Fu97 cell line that was modified to reduce the secretion of TGFβ1,reduce the expression of CD276, and to express GM-CSF, membrane boundCD40L, and IL-12 was also transduced with lentiviral particlesexpressing the modWT1 and modCLDN18 antigens (SEQ ID NO: 55, SEQ ID NO:56). Expression of modWT1 by Fu97 was characterized by flow cytometry.Unmodified and antigen modified cells were stained intracellular with0.03 μg/test anti-rabbit IgG1 anti-WT1 antibody (Abcam, ab89901)followed by 0.125 ug/test AF647-conjugated donkey anti-rabbit IgG1antibody (BioLegend #406414). Expression of modWT1 increased in themodified cell line (7,418,365 MFI) 57-fold over that of the unmodifiedcell line (129,611 MFI) (FIG. 117C). Expression of modCLDN18 by Fu97 wascharacterized by flow cytometry. Unmodified and antigen modified cellswere stained intracellular with 0.03 μg/test anti-rabbit IgG1anti-CLDN18 antibody (Abcam, ab203563) followed by 0.125 ug/testAF647-conjugated donkey anti-rabbit IgG1 antibody (BioLegend #406414).Expression of modCLDN18 increased in the modified cell line (3,168,563MFI) 5.7-fold over that of the unmodified cell line (558,211 MFI) (FIG.117D).

Immune Responses to PSMA and LY6K in GCA Vaccine-A

IFNγ responses to PSMA and LY6K were evaluated in the context of GCAvaccine-A as described in Example 29 and herein, in six HLA diversedonors (n=4/donor). The HLA-A, HLA-B, and HLA-C alleles for each of thesix donors are shown in Table 108. IFNγ responses were determined byELISpot as described in Example 29.

PSMA specific IFNγ responses were increased with the modified GCAvaccine-A (2,413±829 SFU) compared to the parental, unmodified GCAvaccine-A (137±82 SFU (FIG. 117E). IFNγ responses to LY6K weredetermined by ELISpot using 15-mers peptides overlapping by 9 aminoacids spanning the entire length of the native LY6K antigen purchasedfrom Thermo Scientific Custom Peptide Service. IFNγ responses to LY6Ksignificantly increased with the modified GCA vaccine-A (1,598±639 SFU)compared to the unmodified GCA vaccine-A (63±30 SFU) (p=0.002,Mann-Whitney U test) (n=6) (FIG. 117F).

Immune Responses to WT1 and CLDN18 in GCA Vaccine-B

IFNγ responses to WT1 and CLDN18 were evaluated in the context ofGCA-vaccine B as described in Example 29 and herein, in six HLA diversedonors (n=4/donor) (Table 108). IFNγ responses against WT1 and CLDN18were determined by ELISpot using 15-mers peptides overlapping by 9 aminoacids spanning the entire length of the native antigen protein purchasedfrom Thermo Scientific Custom Peptide Service. WT1 specific IFNγresponses increased with GCA vaccine-B (686±330 SFU) compared to theunmodified GCA vaccine-B (37±22 SFU) (n=6) (FIG. 117G). CLDN18 specificIFNγ responses were significantly increased by GCA vaccine-B (1,682±773SFU) compared to the unmodified GCA vaccine-B (113±65 SFU) (p=0.026,Mann-Whitney U test) (n=6) (FIG. 117H).

TABLE 108 Healthy Donor MHC-I characteristics Donor # HLA-A HLA-B HLA-C1 *02:01 *02:01 *15:01 *51:01 *02:02 *03:04 2 *01:01 *32:01 *08:01*14:01 *07:01 *08:01 3 *03:01 *25:01 *07:02 *18:01 *07:02 *12:03 4*02:01 *30:02 *14:02 *57:02 *08:02 *18:02 5 *02:01 *33:01 *07:02 *14:02*07:02 *08:02 6 *01:01 *32:01 *35:01 *40:06 *04:01 *15:02

Cocktails Induce Immune Responses Against Relevant TAAs

The ability of GCA vaccine-A and GCA vaccine-B to induce IFNγ productionagainst ten GCA antigens was measured by ELISpot. PBMCs from sevenHLA-diverse healthy donors (Table 108) were co-cultured with autologousDCs loaded with GCA vaccine-A or GCA vaccine-B for 6 days prior tostimulation with TAA-specific specific peptide pools containing knownMHC-I restricted epitopes. Peptides for stimulation of CD14-PBMCs todetect IFNγ responses to PSMA, LY6K, WT1 and CLDN18 are described above.Additional 15-mer peptides overlapping by 11 amino acid peptide poolswere sourced as follows: MSLN (GenScript custom peptide library), MAGEA3(JPT, PM-MAGEA3), CEA (JPT, PM-CEA), Survivin (thinkpeptides,7769_001-011), STEAP1 (PM-STEAP1) and MUC1 (JPT, PM-MUC1).

FIG. 119 demonstrates the GCA vaccine is capable of inducing antigenspecific IFNγ responses in six HLA-diverse donors to ten GCA antigensthat are 17.5-fold more robust (32,898±13,617 SFU) compared to theunmodified parental control (1,879±463 SFU) (p=0.009, Mann-Whitney Utest) (n=6) (FIG. 119A) (Table 109). The unit dose of GCA vaccine-A andGCA vaccine-B elicited IFNγ responses to eight antigens in two donors,nine antigens in one donor and ten antigens in four donors (FIG. 120).GCA vaccine-A and GCA vaccine-B independently demonstrated a 17.4-foldand 17.7-fold increase antigen specific responses compared to parentalcontrols, respectively. Specifically, GCA vaccine-A elicited18,332±6,823 SFU compared to the unmodified controls (1,055±518 SFU)(p=0.004, Mann-Whitney U test) (FIG. 119B). For GCA vaccine-A, one donorresponded to five antigens, two donors responded to nine antigens, andthree donors responded ten antigens. GCA vaccine-B elicited 14,566±7,499SFU compared to parental controls (823±287 SFU) (p=0.015, Mann-Whitney Utest) (FIG. 119C). For GCA vaccine-B, one donor responded to fiveantigens, two donors responded to nine antigens, and three donorsresponded ten antigens. Described above are two compositions comprisinga therapeutically effective amount of three cancer cell lines, a unitdose of six cell lines, wherein said unit dose is capable of elicitingan immune response 17.5-fold greater than the unmodified compositionspecific to at least eight TAAs expressed in GCA patient tumors. GCAvaccine-A increased IFNγ responses to at least five TAAs 17.4 and GCAvaccine-B increased IFNγ responses 17.7 to at least five TAAs.

TABLE 109 IFNγ Responses to unmodified and modified GCA vaccinecomponents Unmodified (SFU ± SEM) Modified (SFU ± SEM) Donor GCA GCA GCAGCA GCA GCA (n = 4) Vaccine-A Vaccine-B Vaccine Vaccine-A Vaccine-BVaccine 1 305 ± 107 73 ± 73 378 ± 173 5,616 ± 1,720 5,438 ± 3,569 11,054± 4,736  2 3,542 ±     24 ± 24 3,566 ± 1,193 35,007 ± 15,203 51,023 ±17,176 86,030 ± 32,196 3 811 ± 119 1,366 ± 468   2,176 ± 531   25,519 ±11,590 11,586 ± 7,416  37,105 ± 18,093 4 0 ± 0 1,313 ± 533   1,313 ±533   1,869 ± 632   675 ± 236 2,544 ± 673   5 530 ± 215 400 ± 173 1,240± 329   32,064 ± 1,785  9,261 ± 3,145 41,326 ± 2,571  6 968 ± 236 1,631± 701   2,599 ± 927   2,920 ± 1,014 4,743 ± 1,593 10,218 ± 585  

Based on the disclosure and data provided herein, a whole cell vaccinefor Gastric Cancer comprising the six cancer cell lines, sourced fromATCC or JCRB, MKN-1 (JCRB, JCRB0252), MKN-45 (JCRB, JCRB0254), MKN-74(JCRB, JCRB0255), OCUM-1 (JCRB, JCRB0192), Fu97 (JCRB, JCRB1074) and DMS53 (ATCC, CRL-2062) is shown in Table 110. The cell lines represent fivegastric cancer cell lines and one small cell lung cancer (SCLC) cellline (DMS 53 ATCC CRL-2062). The cell lines have been divided into twogroupings: vaccine-A and vaccine-B. Vaccine-A is designed to beadministered intradermally in the upper arm and vaccine-B is designed tobe administered intradermally in the thigh. Vaccine A and B togethercomprise a unit dose of cancer vaccine.

TABLE 110 Cell line nomenclature and modifications TGFβ2 CD276 CocktailCell Line TGFβ1 KD KD KO GM-CSF CD40L IL-12 TAA(s) A MKN-1 X X X X X X XA MKN-45* X ND X X X X ND A MKN-74 X ND X X X X ND B OCUM-1* ND ND X X XND ND B Fu97 X ND X X X X X B DMS 53* ND X X X X ND ND ND = Not done.*Cell lines identified as CSC-like cells.

Where indicated in the above table, the genes for the immunosuppressivefactors transforming growth factor-beta 1 (TGFβ1) and transforminggrowth factor-beta 2 (TGFβ2) have been knocked down using shRNAtransduction with a lentiviral vector. The gene for CD276 has beenknocked out by electroporation using zinc-finger nuclease (ZFN) orknocked down using shRNA transduction with a lentiviral vector. Thegenes for granulocyte macrophage—colony stimulating factor (GM-CSF),IL-12, CD40L, modPSMA (MKN-1), modLY6K (MKN-1), modWT1 (Fu97) andmodCLDN18 (Fu97) have been added by lentiviral vector transduction.

Provided herein are two compositions comprising a therapeuticallyeffective amount of three cancer cell lines, a unit dose of six cancercell lines, modified to reduce the expression of at least oneimmunosuppressive factor and to express at least two immunostimulatoryfactors. One composition, GCA vaccine-A, was modified to increase theexpression of two TAAs, modPSMA and modLY6K. The second composition, GCAvaccine-B, was modified to expresses two TAAs, modWT1 and modCLDN18. Theunit dose of six cancer cell lines expresses at least at least 11 TAAsassociated with a cancer of a subset of gastric cancer subjects intendedto receive said composition and induces IFNγ responses 17.5-fold greaterthan the unmodified composition components.

Example 36: Preparation of Breast Cancer (BRCA) Vaccine

This Example demonstrates that reduction of TGFβ1, TGFβ2, and CD276expression with concurrent overexpression of GM-CSF, CD40L, and IL-12 ina vaccine composition of two cocktails, each cocktail composed of threecell lines for a total of 6 cell lines, significantly increased themagnitude of cellular immune responses to at least 10 BRC-associatedantigens in an HLA-diverse population. As described herein, the firstcocktail, BRC vaccine-A, is composed of cell line CAMA-1 that was alsomodified to express modPSMA, cell line AU565 that was also modified toexpress modTERT, and cell line HS-578T. The second cocktail, BRCvaccine-B, is composed of cell line MCF-7, cell line T47D that was alsomodified to express modTBXT and modBORIS, and cell line DMS 53. The sixcomponent cell lines collectively express at least twenty-two antigensthat can provide an anti-BRC tumor response.

Identification of BRC Vaccine Components

Initial cell line selection criteria identified twenty-nine vaccinecomponent cell lines for potential inclusion in the BRC vaccine.Additional selection criteria described herein were applied to narrowthe twenty-nine cell lines to seven cell lines for further evaluation inimmunogenicity assays. These criteria included: endogenous BRCassociated antigen expression, endogenous expression of antigensenriched in triple negative breast cancer, lack of expression ofadditional immunosuppressive factors, such as IL-10 or IDO1, expressionof BRC-associated CSC-like markers ABCG2, ALDH1A, BMI1, CD133, CD44,ITGA6, CD90, c-myc, CXCR1 CXCR4, EPCAM, KLF4, MUC1, NANOG, SAL4 andSOX2, ethnicity and age of the patient from which the cell line wasderived, site and stage of the breast cancer, molecular subtype andhistological subtype.

CSCs play a critical role in the metastasis, treatment resistance, andrelapse of breast cancer (Table 2). Expression of TAAs and BRC specificCSC-like markers by candidate component cell lines was determined by RNAexpression data sourced from the Broad Institute Cancer Cell LineEncyclopedia (CCLE). The HGNC gene symbol was included in the CCLEsearch and mRNA expression was downloaded for each TAA. Expression of aTAA or CSC marker by a cell line was considered positive if the RNA-seqvalue was greater than one. Selection criteria identified sevencandidate BRC vaccine components for further evaluation: BT20, HS-578T,AU565, ZR751, MCF-7, CAMA-1 and T47D. The seven candidate component celllines expressed seven to eleven TAAs (FIG. 121A) and six to nine CSCmarkers (FIG. 121B). As described herein, the CSC-like cell line DMS 53is included as one of the six vaccine cell lines and expressed fifteenBRC TAAs and three BRC CSC-like markers.

Immunogenicity of the seven unmodified BRC vaccine component candidateswere evaluated by IFNγ ELISpot as described in Example 9 using three HLAdiverse healthy donors (n=4 per donor). HLA-A and HLA-B alleles forDonor 1 were A*02:01 B*57:03 and A*01:01 B*08:01. HLA-A and HLA-Balleles for Donor 2 were A*30:01 B*57:01 and A*02:01 B*40:01. HLA-Aalleles for Donor 3 were A*01:01 and A*02:01. HLA-B typing was notavailable for Donor 3. Immunogenicity of T47D was evaluated separatelyin five HLA diverse donors (Table 117, Donors 2-6). MCF-7 (2,314±448SFU) and CAMA-1 (990±223 SFU) were more immunogenic than AU565 (274±87SFU), ZR751 (292±133 SFU), BT20 (524±192 SFU), HS-578T (281±81 SFU)(FIG. 122A) and T47D (491±202 SFU) (FIG. 122C).

Immunogenicity of MCF-7 and CAMA-1 were evaluated in eight differentcombinations of three component cell lines, four combinations containedMCF-7 and four combinations contained CAMA-1 (FIG. 122D). IFNγ responseswere determined against the three component cell lines within the eightpotential vaccine cocktails by IFNγ ELISpot as described in Example 8using the three healthy donors (n=4/donor). HLA-A and HLA-B alleles forthe Donors were as follows: Donor 1, A*01:01 B*08:01 and A*02:01B*15:01; Donor 2, A*03:01 B*15:01 and A*24:02 B*07:02; Donor 3, A*01:01B*30:01 and A*02:01 B*12:02. One additional cocktail combination ofthree component cell lines including T47D, MCF-7 and DMS 53 T47D wasalso evaluated (FIG. 122C) in the same five HLA-diverse donors (Table117, Donors 2-6). IFNγ responses were detected for all nine cocktailsand to each cell line component in each cocktail.

In all eight combinations evaluated, MCF-7 and CAMA-1 remained the mostimmunogenic. Responses to the individual cocktail component cell lineswere similar, except for CAMA-1 and ZR751. IFNγ responses to CAMA-1slightly decreased in the three component cell line combinations. IFNγresponses to ZR751 also slightly decreased in the three cell linecomponent cocktails and therefore ZR751 was not included in the BRCvaccine (FIG. 122B-C). Triple negative breast cancer comprisesapproximately 15% of breast cancers. For this reason, one triplenegative breast cancer cell line, 17% of the unit dose of the BRCvaccine, was included in the composition vaccine. The immunogenicity ofthe triple negative breast cancer cell lines, BT20 and HS-578T, wassimilar when evaluated in three cell line component cocktails. Of thesetwo cell lines, HS-578T endogenously expressed more TAAs (elevenTAAs>1.0 FPKM) than BT20 (nine TAAs>1.0 FPKM) (FIG. 121A) and wasselected for inclusion in the BRC vaccine. CAMA-1 was selected to beincluded in vaccine cocktail A and MCF-7 selected to be included invaccine cocktail B as described above and further herein.

The cells in the vaccine described herein were selected to express awide array of TAAs, including those known to be important specificallyfor BRC antitumor responses, such as mammaglobin A (SCGB2A2) and MUC1,enriched in triple negative breast cancer, such as TBXT and NY-ESO-1,and also TAAs known to be important for targets for BRC and other solidtumors, such TERT. As shown herein, to further enhance the array ofTAAs, CAMA-1 was modified to express modPSMA, AU565 was modified toexpress modTERT, and T47D that was also modified to express modTBXT andmodBORIS.

TBXT and BORIS were not endogenously expressed in any of the sixcomponent cell lines at >1.0 FPKM. TERT and PSMA were endogenouslyexpressed by one of the six component cell lines at >1.0 FPKM (FIG.123A).

Expression of the transduced antigens modPSMA (FIG. 124A) by CAMA-1 (SEQID NO: 37; SEQ ID NO: 38), modTERT (FIG. 124B) by AU565 (SEQ ID NO: 35;SEQ ID NO: 36), and modTBXT (FIG. 124C) and modBORIS (FIG. 124D) (SEQ IDNO: 41; SEQ ID NO: 42) by T47D, were detected by flow cytometry orRT-PCR as described in Example 29 and herein. The modTBXT and modBORISantigens are encoded in the same lentiviral transfer vector separated bya furin cleavage site (SEQ ID NO: 41 and SEQ ID NO: 42).

Because of the need to maintain maximal heterogeneity of antigens andclonal subpopulations the comprise each cell line, the gene modifiedcell lines utilized in the present vaccine have been established usingantibiotic selection and flow cytometry and not through limitingdilution subcloning.

The endogenous mRNA expression of twenty-two representative BRC TAAs inthe present vaccine are shown in FIG. 123A. The present vaccine, afterintroduction of the antigens described above, expresses of allidentified twenty-two commonly targeted and potentially clinicallyrelevant TAAs capable of inducing a BRC antitumor response. Some ofthese TAAs are known to be primarily enriched in BRC tumors and some canalso induce an immune response to BRC and other solid tumors. RNAabundance of the twenty-two prioritized BRC TAAs was determined in 1082BRC patient samples with available mRNA data expression as described inExample 29 (FIG. 123B). Fifteen of the prioritized BRC TAAs wereexpressed by 100% of samples, 16 TAAs were expressed by 99.9% ofsamples, 17 TAAs were expressed by 99.3% of samples, 18 TAAs wereexpressed by 95.1% of samples, 19 TAAs were expressed by 79.9% ofsamples, 20 TAAs were expressed by 47.6% of samples, 21 TAAs wereexpressed by 17.1% of samples, and 22 TAAs were expressed by 3.4% ofsamples (FIG. 123C). Provided herein are two compositions comprising atherapeutically effective amount of three cancer cell lines, wherein thecombination of the cell lines, a unit dose of six cell lines, comprisescells that express at least 15 TAAs associated with a subset of BRCcancer subjects intended to receive said composition. Based on theexpression and immunogenicity data presented herein, the cell linesidentified in Table 111 were selected to comprise the present BRCvaccine.

TABLE 111 Breast vaccine cell lines and histology Cocktail Cell LineName Histology A CAMA-1 Breast Luminal A Adenocarcinoma, ER+, PR+,Her2−; derived from metastatic site (pleural effusion) A AU565 BreastLuminal Adenocarcinoma, ER−, PR−, Her2+; derived from metastatic site(pleural effusion) A HS-578T Breast Triple Negative Ductal Carcinoma,ER−, PR−, Her2− B MCF-7 Breast Luminal A Adenocarcinoma, ER+, PR+, Her2;derived from metastatic site (pleural effusion) B T47D Breast Luminal ADuctal Carcinoma, ER+, PR+, Her2; derived from metastatic site (pleuraleffusion) B DMS 53 Lung Small Cell Carcinoma

Reduction of CD276 Expression

The CAMA-1, AU565, HS-578T, MCF-7, T47D, and DMS 53 component cell linesexpressed CD276 and expression was knocked out by electroporation withZFN as described in Example 13 and elsewhere herein. Because it wasdesirable to maintain as much tumor heterogeneity as possible, theelectroporated and shRNA modified cells were not cloned by limitingdilution. Instead, the cells were subjected to multiple rounds of cellsorting by FACS as described in Example 13. Expression of CD276 wasdetermined as described in Example 29. Reduction of CD276 expression isdescribed in Table 112. These data show that gene editing of CD276 withZFN resulted in greater than 95.2% CD276-negative cells in all sixvaccine component cell lines.

TABLE 112 Reduction of CD276 expression Parental Modified % ReductionCell line Cell Line MFI Cell Line MFI CD276 CAMA-1 14,699 75 99.5 AU5654,085 0 ≥99.9 HS-578T 33,832 234 99.3 MCF-7 25,952 1,243 95.2 T47D11,737 3 ≥99.9 DMS 53 11,928 24 99.8 MFI reported with isotype controlssubtracted

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12 werecompleted as described in Example 29.

shRNA Downregulates TGF-β Secretion

Following CD276 knockout, TGFβ1 and TGFβ2 secretion levels were reducedusing shRNA and resulting levels determined as described in Example 29.The AU565 and HS-578T parental cell lines in BRC vaccine-A secretedmeasurable levels of TGFβ1 and TGFβ2. CAMA-1 secreted detectable levelsof TGFβ2 but not TGFβ1. The MCF-7 component cell line of BRC vaccine-Bsecreted measurable levels of TGFβ1 and TGFβ2. T47D did not secretedmeasurable levels of TGFβ1 or TGFβ2 and therefore was not modified toreduce secretion of TGFβ1 or TGFβ2. Reduction of TGFβ2 secretion by theDMS 53 cell line is described in Example 26 and resulting levelsdetermined as described above and herein.

The component HS-578T and MCF-7 cell lines were transduced with TGFβ1shRNA to decrease TGFβ1 secretion concurrently with the transgene toincrease expression of membrane bound CD40L as described in Example 29.HS-578T and MCF-7 were also transduced with lentiviral particlesencoding TGFβ2 shRNA to decrease the secretion of TGFβ2 and concurrentlyincrease expression of GM-CSF (SEQ ID NO: 6) as described in Example 29.These cells are described by the clonal designation DK6. The HS-578T,MCF-7, CAMA-1 and AU565 cell lines were transduced with lentiviralparticles encoding TGFβ2 shRNA to decrease the secretion of TGFβ2 andconcurrently increase expression of GM-CSF (SEQ ID NO: 6) as describedin Example 29. DMS 53 was modified with shRNA to reduce secretion ofTGFβ2 as described in Example 26. The cell lines modified to reducesecretion of TGFβ2 and not TGFβ1 are described by the clonal designationDK4.

Table 113 shows the percent reduction in TGFβ1 and/or TGFβ2 secretion ingene modified component cell lines compared to unmodified, parental celllines. If TGFβ1 or TGFβ2 secretion was only detected in 1 of 16replicates run in the ELISA assay the value is reported without standarderror of the mean. Gene modification resulted at least 44% reduction ofTGFβ1 secretion. Gene modification of TGFβ2 resulted in at least 51%reduction in secretion of TGFβ2.

TABLE 113 TGF-β Secretion (pg/10⁶ cells/24 hr) in Component Cell LinesCell Line Cocktail Clone TGFβ1 TGFβ2 CAMA-1 A Wild type * ≤20 249 ± 59 CAMA-1 A DK4 NA * ≤11 CAMA-1 A Percent reduction 79% 96% AU565 A Wildtype 325 ± 219 306 ± 294 AU565 A DK4 NA * ≤23 AU565 A Percent reduction≥85%   ≥92%   HS-578T A Wild type 3,574 ± 690   615 ± 247 HS-578T A DK61,989 ± 200   118 ± 26  HS-578T A Percent reduction 44% 81% MCF-7 B Wildtype 1,279 ± 174   411 ± 149 MCF-7 B DK6 306 ± 48  * ≤14 MCF-7 B Percentreduction 76% 60% T47D B Wild type * ≤32 * ≤15 T47D B NA NA NA T47D BPercent reduction NA NA DMS 53 B Wild type 106 ± 10  486 ± 35  DMS 53 BDK4 NA 238 ± 40  DMS 53 B Percent reduction NA 51% DK6: TGFβ1/TGFβ2double knockdown; DK4: TGFβ2 single knockdown; DK2: TGFβ1 singleknockdown; * = estimated using LLD, not detected; NA = not applicable

Based on a dose of 5×10⁵ of each component cell line, the total TGFβ1and TGFβ2 secretion by the modified BRC vaccine-A and BRC vaccine-B andrespective unmodified parental cell lines are shown in Table 114. Thesecretion of TGFβ1 by BRC vaccine-A was reduced by 49% and TGFβ2 by 87%pg/dose/24 hr. The secretion of TGFβ1 by BRC vaccine-B was reduced by67% and TGFβ2 by 71% pg/dose/24 hr.

TABLE 114 Total TGF-β Secretion (pg/dose/24 hr) in BRC vaccine-A and BRCvaccine-B Cocktail Clones TGFβ1 TGFβ2 A Wild type 1,960 585 DK4/DK6 99576 Percent reduction 49% 87% B Wild type 709 456 DK4/DK6 222 134 Percentreduction 67% 71%

GM-CSF Secretion

The HS-578T, MCF-7, CAMA-1 and AU565 cell lines were transduced withlentiviral particles containing both TGFβ2 shRNA and the gene to expressGM-CSF (SEQ ID NO: 6) as described above. The T47D cell line wastransduced with lentiviral particles to only express GM-CSF (SEQ ID NO:7). DMS 53 was modified to secrete GM-CSF as described in Example 26 andelsewhere herein. The results are shown in Table 115 and describedbelow.

Secretion of GM-CSF increased at least 15,714-fold in all modifiedcomponent cell lines compared to unmodified, parental cell lines. In BRCvaccine-A component cell lines, secretion of GM-CSF increased36,990-fold by CAMA-1 compared to the parental cell line (≤0.0039 ng/10⁶cells/24 hr), 15,714-fold by AU565 compared to the parental cell line(≤0.0042 ng/10⁶ cells/24 hr), and 21,061-fold by HS-578T compared to theparental cell line (≤0.0064 ng/10⁶ cells/24 hr). In BRC vaccine-Bcomponent cell lines secretion of GM-CSF increased 25,528-fold by MCF-7compared to the parental cell line (≤0.0118 ng/10⁶ cells/24 hr),33,920-fold by T47D compared to the parental cell line (≤0.0063 ng/10⁶cells/24 hr) and 49,313-fold by DMS 53 compared to the parental cellline (≤0.0032 ng/10⁶ cells/24 hr).

TABLE 115 GM-CSF Secretion in Component Cell Lines GM-CSF GM-CSF CellLine (ng/10⁶ cells/24 hr) (ng/dose/24 hr) CAMA-1 145 ± 30  73 AU565 66 ±37 33 HS-578T 135 ± 20  68 Cocktail A Total 346 174 MCF-7 302 ± 66  151T47D 212 ± 40  106 DMS 53 158 ± 15  79 Cocktail B Total 672 336

Based on a dose of 5×10⁵ of each component cell line, the total GM-CSFsecretion for BRC vaccine-A was 174 ng per dose per 24 hours. The totalGM-CSF secretion for BRC vaccine-B was 336 ng per dose per 24 hours. Thetotal GM-CSF secretion per dose was therefore 510 ng per 24 hours.

Membrane Bound CD40L (CD154) Expression

The component cell lines were transduced with lentiviral particles toexpress membrane bound CD40L vector as described above. The methods todetect expression of CD40L by the five BRC cell line components aredescribed in Example 29. Modification of DMS 53 to express membranebound CD40L is described in Example 15. Evaluation of membrane boundCD40L by all six vaccine component cell lines is described below. Theresults shown in FIG. 125 and described below demonstrate CD40L membraneexpression was substantially increased in all six BRC vaccine componentcell lines.

Expression of membrane bound CD40L increased at least 3,417-fold in allcomponent cell lines compared to unmodified, parental cell lines. In BRCvaccine-A component cell lines, expression of CD40L increased 3,417-foldby CAMA-1 (3,417 MFI) compared to the parental cell line (0 MFI),6,527-fold by AU565 (6,527 MFI) compared to the parental cell line (0MFI), and 6,560-fold by HS-578T (6,560 MFI) compared to the parentalcell line (0 MFI). In BR-BT vaccine-B component cell lines expression ofCD40L increased 5,986-fold by MCF-7 (5,986 MFI) compared to the parentalcell line (0 MFI), 45,071-fold by T47D (45,071 MFI) compared to theparental cell line (0 MFI), and 88,261-fold by DMS 53 compared to theparental cell line (0 MFI).

IL-12 Expression

The component cell lines were transduced with the IL-12 vector asdescribed in Example 17 and resulting IL-12 p70 expression determined asdescribed above and herein. The results are shown in Table 116 anddescribed below.

Secretion of IL-12 increased at least 4,034-fold in all component celllines modified to secrete IL-12 p70 compared to unmodified, parentalcell lines. In BRC vaccine-A component cell lines, secretion of IL-12increased 39,490-fold by CAMA-1 compared to the parental cell line(≤0.0016 ng/10⁶ cells/24 hr), 14,793-fold by AU565 compared to theparental cell line (≤0.0017 ng/10⁶ cells/24 hr), and 19,141-fold byHS-578T compared to the parental cell line (≤0.0026 ng/10⁶ cells/24 hr).In BRC vaccine-B component cell lines expression of IL-12 increased4,034-fold by MCF-7 compared to the parental cell line (≤0.0047 ng/10⁶cells/24 hr) and 43,655-fold by T47D compared to the parental cell line(≤0.002 ng/10⁶ cells/24 hr). DMS 53 was not modified to secrete IL-12.

TABLE 116 IL-12 Secretion in Component Cell Lines IL-12 IL-12 Cell Line(ng/10⁶ cells/24 hr) (ng/dose/24 hr) CAMA-1 62 ± 13 31 AU565 25 ± 12 13HS-578T 49 ± 11 25 Cocktail A Total 136 69 MCF-7 19 ± 13 10 T47D 86 ± 1743 DMS 53 NA NA Cocktail B Total 105 53

Based on a dose of 5×10⁵ of each component cell line, the total IL-12secretion for BRC vaccine-A was 69 ng per dose per 24 hours. The totalIL-12 secretion for BRC vaccine-B was 53 ng per dose per 24 hours. Thetotal IL-12 secretion per dose was therefore 122 ng per 24 hours.

Stable Expression of modPSMA by the CAMA-1 Cell Line

As described above, the cells in the vaccine described herein wereselected to express a wide array of TAAs, including those known to beimportant to antitumor immunity. To further enhance the array ofantigens, the CAMA-1 cell line that was modified to reduce the secretionof TGFβ2, reduce the expression of CD276, and to express GM-CSF,membrane bound CD40L and IL-12 was also transduced with lentiviralparticles expressing the modPSMA antigen (SEQ ID NO: 37, SEQ ID NO: 38).The expression of modPSMA by CAMA1 was characterized by flow cytometry.Unmodified and antigen modified cells were stained intracellular with0.03 μg/test anti-mouse IgG1 anti-PSMA antibody (Abcam, ab268061)followed by 0.125 ug/test AF647-conjugated goat anti-mouse IgG1 antibody(BioLegend #405322). Expression of modPSMA was increased in the modifiedcell line (77,718 MFI) 17-fold over that of the parental cell line(4,269 MFI) (FIG. 124A).

Stable Expression of modTERT by the AU565 Cell Line

The AU565 cell line that was modified to reduce the secretion of TGFβ2,reduce the expression of CD276, and to express GM-CSF, membrane boundCD40L and IL-12 was also transduced with lentiviral particles expressingthe modTERT antigen (SEQ ID NO: 35, SEQ ID NO: 36). Expression ofmodTERT by AU565 was characterized by flow cytometry. Unmodified andantigen modified cells were stained intracellular with 0.03 μg/testanti-mouse IgG1 anti-TERT antibody (Abcam, ab32020) followed by 0.125ug/test donkey anti-rabbit IgG1 antibody (BioLegend #406414). Expressionof modTERT was increased in the modified cell line (957,873 MFI) 31-foldover that of the unmodified cell line (30,743 MFI) (FIG. 124B).

Stable Expression of modTBXT and modBORIS by the T47D Cell Line

The T47D cell line that was modified to reduce the reduce the expressionof CD276, and to express GM-CSF, membrane bound CD40L, and IL-12 wasalso transduced with lentiviral particles expressing the modTBXT andmodBORIS antigens (SEQ ID NO: 41, SEQ ID NO: 42). Expression of modTBXTby T47D was characterized by flow cytometry. Unmodified and antigenmodified cells were stained intracellular with 0.06 μg/test anti-rabbitIgG1 anti-TBXT antibody (Abcam, ab209665) followed by 0.125 ug/testAF647-conjugated donkey anti-rabbit IgG1 antibody (BioLegend #406414).Expression of modTBXT increased in the modified cell line (147,610 MFI)147,610-fold over that of the unmodified cell line (0 MFI) (FIG. 124C).Expression of BORIS by SCaBER was determined by RT-PCR as described inExample 29 and herein. The forward primer was designed to anneal at the1119-1138 bp location in the transgene (TTCCAGTGCTGCCAGTGTAG (SEQ IDNO:134)) and reverse primer designed to anneal at the 1159-1178 bplocation in the transgene (AGCACTTGTTGCAGCTCAGA (SEQ ID NO: 135))yielding a 460 bp product. Control primers for β-tubulin are describedin Example 29. The gene product for modBORIS was detected at theexpected size (FIG. 124D) and mRNA increased 2,198-fold relative to theparental control.

Immune Responses to PSMA in BRC Vaccine-A

IFNγ responses to PSMA were evaluated in the context of BRC vaccine-A asdescribed in Example 29, and herein, in six HLA diverse donors(n=4/donor). The HLA-A, HLA-B, and HLA-C alleles for each of the sixdonors are shown in Table 117. IFNγ responses were determined by ELISpotas described in Example 29 using 15-mers peptides overlapping by 9 aminoacids spanning the entire length of the native PSMA antigen purchasedfrom Thermo Scientific Custom Peptide Service. PSMA specific IFNγresponses with the were significantly increased with the modified BRCvaccine-A (4,166±1,647 SFU) compared to the parental, unmodified BRCvaccine-A (393±210 SFU (p=0.041, Mann-Whitney U test) (n=6) (FIG. 124E).

Immune Responses to TERT in BRC Vaccine-A

IFNγ responses to TERT were evaluated in the context of BRC vaccine-A asdescribed in Example 29, and herein, in six HLA diverse donors(n=4/donor). The HLA-A, HLA-B, and HLA-C alleles for each of the sixdonors are shown in Table 117. IFNγ responses were determined by ELISpotusing 15-mers peptides overlapping by 11 amino acids spanning the entirelength of the native TERT antigen (JPT, PM-TERT). IFNγ responses to TERTincreased with the modified BRC vaccine-A (3,807±927 SFU) compared tothe unmodified BRC vaccine-A (1,670±918) SFU (FIG. 124F).

Immune Responses to TBXT and BORIS in BRC Vaccine-B

IFNγ responses to TBXT and BORIS were evaluated in the context ofBRC-vaccine B as described in Example 32, and herein, in six HLA diversedonors (n=4/donor) (Table 117). IFNγ responses against TBXT weredetermined by ELISpot using 15-mers peptides overlapping by 11 aminoacids spanning the entire length of the native antigen (JPT, PM-BRAC).IFNγ responses against BORIS were determined by ELISpot using 15-merspeptides overlapping by 9 amino acids spanning the entire length of thenative antigen protein purchased from Thermo Scientific Custom PeptideService.

TBXT specific IFNγ responses were increased by BRC vaccine-B (1,102±366SFU) compared to the unmodified BRC vaccine-B (930±496 SFU) (n=6) (FIG.124G). BORIS specific IFNγ responses were also increased by BRCvaccine-B (3,054±1,155 SFU) compared to the unmodified BRC vaccine-B(1,757±661 SFU) (n=6) (FIG. 124H).

TABLE 117 Healthy Donor MHC-I characteristics Donor # HLA-A HLA-B HLA-C1 *03:01 *07:02 *07:02 *35:01 *04:01 *07:02 2 *02:01 *03:01 *27:05*27:05 *01:02 *01:02 3 *02:01 *33:01 *07:02 *14:02 *07:02 *08:02 4*02:01 *02:01 *15:01 *44:02 *03:04 *14:02 5 *24:02 *02:01 *08:01 *51:01*14:02 *03:04 6 *01:01 *02:01 *35:01 *50:01 *04:01 *06:02

Cocktails Induce Immune Responses Against Relevant TAAs

The ability of BRC vaccine-A and BRC vaccine-B to induce IFNγ productionagainst ten BRC antigens was measured by ELISpot. PBMCs from sixHLA-diverse healthy donors (Table 117) were co-cultured with autologousDCs loaded with BRC vaccine-A or BRC vaccine-B for 6 days prior tostimulation with TAA-specific specific peptide pools containing knownMHC-I restricted epitopes. Peptides for stimulation of CD14-PBMCs todetect IFNγ responses to PSMA, TERT, TBXT and BORIS are described above.Additional 15-mer peptides overlapping by 11 amino acid peptide poolswere sourced as follows: STEAP1 (PM-STEAP1), PRAME (JPT, PM-01P4),SCGB2A2 (Mammaglobin-A) (JPT, PM-MamA), Survivin (thinkpeptides,7769_001-011), MUC1 (JPT, PM-MUC1) and MMP11 (JPT, PM-MMP11).

FIG. 126 demonstrates the BRC vaccine is capable of inducing antigenspecific IFNγ responses in six HLA-diverse donors to ten BRC antigensthat are 2.2-fold more robust (45,370±9,212 SFU) compared to theunmodified parental control (20,183±7,978 SFU) (n=6) (FIG. 136A) (Table118). The unit dose of BRC vaccine-A and BRC vaccine-B elicited IFNγresponses to nine antigens in two donors and ten antigens in four donors(FIG. 127). The BRC vaccine increase IFNγ responses to PRAME 2.2-fold(3,049±1,079 SFU) and TBXT 1.7-fold, (3,049±1,079 SFU), two antigensenriched in the triple negative molecular subset of breast cancer,compared to the unmodified controls, 1,380±697 SFU and 1,601±810 SFU,respectively. BRC vaccine-A and BRC vaccine-B independently demonstrateda 2.6-fold and 1.4-fold increase antigen specific responses compared toparental controls, respectively. Specifically, BRC vaccine-Asignificantly increase antigen specific response 23,944±3,971 SFUcompared to the unmodified controls (9,197±3,433 SFU) (p=0.026,Mann-Whitney U test) (FIG. 127B). For BRC vaccine-A, two donorsresponded to nine antigens and four donors responded ten antigens. BRCvaccine-B elicited 17,032±3,861 SFU compared to parental controls(11,975±4,510 SFU) (n=6) (FIG. 127C). For BRC vaccine-B, one donorresponded to five antigens, two donors responded to nine antigens, andthree donors responded to ten antigens. Described above are twocompositions comprising a therapeutically effective amount of threecancer cell lines, a unit dose of six cell lines, wherein said unit doseis capable of eliciting an immune response 2.2-fold greater than theunmodified composition specific to at least nine TAAs expressed in BRCpatient tumors. BRC vaccine-A increased IFNγ responses to at least nineTAAs 2.6-fold and BRC vaccine-B increased IFNγ responses 1.4-fold to atleast five TAAs.

TABLE 118 IFNγ Responses to unmodified and modified BRC vaccinecomponents Unmodified (SFU ± SEM) Modified (SFU ± SEM) Donor BRC BRC BRCBRC BRC BRC (n = 4) Vaccine-A Vaccine-B Vaccine Vaccine-A Vaccine-BVaccine 1 2,590 ± 924   4,896 ± 2,759 14,248 ± 9,736  41,841 ± 10,93429,895 ± 9,674 71,736 ± 19,975 2 2,134 ± 434   1,697 ± 197   4,061 ±761   36,234 ± 4,700  31,114 ± 1,918 67,349 ± 6,540   3* 4,867 ± 4,50311,522 ± 6,462  19,399 ± 14,052 6,345 ± 3,166  2,802 ± 1,446 12,196 ±4,892  4 9,535 ± 7,710 14,104 ± 8,363  21,073 ± 16,703 23,510 ± 10,746 9,724 ± 5,389 33,234 ± 16,056 5 23,976 ± 17,601 38,089 ± 18,754 57,350± 38,795 17,257 ± 7,954  17,712 ±     36,268 ± 18,735 6 3,397 ± 992  659 ± 331 4,968 ± 2,159 30,599 ± 10,330 20,841 ± 5,625 51,440 ± 15,727*Donor 3 n = 3. All other Donors n = 4.

Cocktails Increase the Breadth and Magnitude of IFNγ Responses to TAAs

The ability of BRC vaccine-A and BRC vaccine-B to elicit a greaterantigenic breadth and magnitude of IFNγ production, as described inExample 8, compared to the single component cell lines, as described inExample 9, was evaluated by IFNγ ELISpot in Donors 2-6 (Table 117). BRCvaccine-A (FIG. 128A) and BRC vaccine-B (FIG. 128B) induced more robustresponses to breast cancer antigens. Importantly, BRC vaccine-A and BRCvaccine-B induced IFNγ responses to a greater number of antigenscompared to single component cell lines. In this subset of five donors,for BRC vaccine-A induced IFNγ responses to nine antigens in two donorsand ten antigens in three donors. CAMA-1 alone induced IFNγ responses tofour antigens in one donor, six antigens in two donors, eight antigensin 1 donor and ten antigens in one donor. AU565 alone induced IFNγresponses to two antigens in one donor, six antigens in two donors, nineantigens in one donor and ten antigens in one donor. HS-578T aloneinduced IFNγ responses to zero antigens in one donor, three antigens inone donor, five antigens in one donor, nine antigens in one donor andten antigens in one donor (FIG. 128C). In this subset of five donors,for BRC vaccine-B induced IFNγ responses to five antigens in one donor,nine antigens in two donors and ten antigens in two donors. MCF-7 aloneinduced IFNγ responses to three antigens in one donor, five antigens inone donor, seven antigens in one donor, eight antigens in 1 donor andten antigens in one donor. T47D alone induced IFNγ responses to zeroantigens in one donor, five antigens in one donor, seven antigens in twodonors, and nine antigens in one donor (FIG. 128D).

Based on the disclosure and data provided herein, a whole cell vaccinefor Breast Cancer comprising the six cancer cell lines, sourced fromATCC, CAMA-1 (ATCC, HTB-21), AU565 (ATCC, CRL-2351), HS-578T (ATCC,HTB-126), MCF-7 (ATCC, HTB-22), T47D (ATCC, HTB-133) and DMS 53 (ATCC,CRL-2062) is shown in Table 119. The cell lines represent five breastcancer cell lines and one small cell lung cancer (SCLC) cell line (DMS53 ATCC CRL-2062). The cell lines have been divided into two groupings:vaccine-A and vaccine-B. Vaccine-A is designed to be administeredintradermally in the upper arm and vaccine-B is designed to beadministered intradermally in the thigh. Vaccine A and B togethercomprise a unit dose of cancer vaccine.

TABLE 119 Cell line nomenclature and modifications TGFβ1 CD276 CocktailCell Line KD TGFβ2 KD KO GM-CSF CD40L IL-12 TAA(s) A CAMA-1 ND X X X X XX A AU565 ND X X X X X X A HS-578T X X X X X X ND B MCF-7 X X X X X X NDB T47D ND ND X X X X X B DMS 53* ND X X X X X ND ND = Not done. *Celllines identified as CSC-like cells.

Where indicated in the above table, the genes for the immunosuppressivefactors transforming growth factor-beta 1 (TGFβ1) and transforminggrowth factor-beta 2 (TGFβ2) have been knocked down using shRNAtransduction with a lentiviral vector. The gene for CD276 has beenknocked out by electroporation using zinc-finger nuclease (ZFN) orknocked down using shRNA transduction with a lentiviral vector. Thegenes for granulocyte macrophage—colony stimulating factor (GM-CSF),IL-12, CD40L, modTERT (AU565), modPSMA (CAMA-1), modTBXT (T47D), andmodBORIS (T47D) have been added by lentiviral vector transduction.

Provided herein are two compositions comprising a therapeuticallyeffective amount of three cancer cell lines, a unit dose of six cancercell lines, modified to reduce the expression of at least oneimmunosuppressive factor and to express at least two immunostimulatoryfactors. One composition, BRC vaccine-A, was modified to increase theexpression of two TAAs, modTERT and modPSMA. The second composition, BRCvaccine-B, was modified to expresses two TAAs, modTBXT and modBORIS. Theunit dose of six cancer cell lines expresses at least at least 15 TAAsassociated with a cancer of a subset of breast cancer subjects intendedto receive said composition and induces IFNγ responses 2.2-fold greaterthan the unmodified composition components.

Example 37: Adaptation of GBM Vaccine Component Cell Lines to Growth inXeno-Free Media

Overview of Adaptation Process

Five component cell lines of the GBM vaccine composition (DBTRG-05MG,LN-229, GB1, KNS-60 and SF-126) were directly cultured in (A1D, A2D) orsequentially adapted (A1W, A2W) to growth in media that is xeno-free,serum-free and devoid of non-human elements. For each cell line, twomedia formulations were tested. Conventional culture media consisted ofRPMI (DBTRG-05MG, LN-229) or DMEM (GB1, KNS-60, SF-126), supplementedwith 10% FBS, L-Glutamine, sodium pyruvate, HEPES, MEM-NEAA(non-essential amino acids used only in DMEM), and antibiotics (Table120). Xeno-free media contained 15% xeno-free replacement (XFR) toreplace FBS, and different antibiotics concentrations than inconventional media (A1-XFR media: RPMI- or DMEM-based media formulatedwith antibiotics shown in Table 121; A2-XFR media: RPMI- or DMEM-basedmedia formulated with antibiotics shown in Table 122). Notably,antibiotics that are added to the media formulation for selection oftransgenes bind to protein present in the media. Due to lower proteinconcentrations in xeno-free media compared to FBS-containing media,antibiotics concentrations were lowered to test two differentconcentrations, respectively, in A1-XFR and A2-XFR media. Each of thefive GBM vaccine component cell lines were screened for growth in 2media formulations A1-XFR and A2-XFR, and two adaptation conditions-comparing direct plating (A1D, A2D) to sequential weaning (A1W, A2W).

To confirm adaptation to xeno-free media formulations, cell morphologyand proliferation were monitored. Culture conditions that showednon-adherent floating cells that were non-viable upon Trypan Bluestaining were terminated. Cell lines with similar morphology to theircontrol in FBS-containing media that were stably growing in xeno-freemedia and were under antibiotic selection for at least 3 weeks wereharvested and analyzed for expression of modified genes.

TABLE 120 Base media (containing FBS) antibiotic concentrations forselection of inserted transgenes Cell Line Blasticidin HygromycinPuromycin DBTRG-05MG 4 300 n/a LN-229 4 300 2 GB1 4 500 n/a KNS-60 4 5002 SF-126 4 500 2 All selection antibiotic concentrations are in μg/mL.n/a, selection antibiotic not used for cell line.

TABLE 121 A1-XFR media antibiotic concentrations for selection ofinserted transgenes Cell Line Blasticidin Hygromycin PuromycinDBTRG-05MG 1.25 100 n/a LN-229 1.25 100 0.4 GB1 1.25 100 n/a KNS-60 1.25100 0.4 SF-126 1.25 100 0.4 All selection antibiotic concentrations arein μg/mL. n/a, selection antibiotic not used for cell line.

TABLE 122 A2-XFR media antibiotic concentrations for selection ofinserted transgenes Cell Line Blasticidin Hygromycin PuromycinDBTRG-05MG 2 200 n/a LN-229 2 200 1 GB1 2 200 n/a KNS-60 2 200 1 SF-1262 200 1 All selection antibiotic concentrations are in μg/mL. n/a,selection antibiotic not used for cell line.

Analysis of Transgene Expression in Cell Lines Grown in Xeno-Free Media

Each of the five modified GBM vaccine component cell lines were screenedfor growth in 2 media formulations A1-XFR and A2-XFR, and two adaptationconditions- comparing direct plating (A1D) to sequential weaning (A1W,A2W). The conditions that showed stable cell growth, minimal cell deathand morphology comparable to cells grown in FBS were analyzed forexpression of transgenes.

To obtain reproducible measurements of secreted cytokines, secretionassays were performed. Cells were seeded in duplicates of 0.7 5×10⁶ and0.5×10⁶ cells per well of a vitronectin-coated 6-well plate in xeno-freemedia. After 24 hours, the media was replaced with fresh xeno-freemedia. After another 48 hours, supernatants were harvested for analysisby ELISA. At the same time, cells were harvested for evaluation of CD40Lexpression by flow cytometry. Briefly, after harvest, cells were stainedwith phycoerythrin-conjugated anti-human CD40L (clone TRAP1). Labelledcells were analyzed by flow cytometry using a LSR Fortessa Flowcytometer. Secreted cytokines were measured using an enzyme linkedimmunosorbent assay (ELISA). Briefly, for each sample, two-fourdilutions of the supernatant were run. TGFβ1 and TGFβ2 levels weredetermined using an enzyme-linked immunosorbent assay (ELISA) (R&DSystems). TGFβ1 and TGFβ2 secretion is reported in units of pg/10⁶cells/24 hours. GM-CSF and IL-12 levels were determined using anenzyme-linked immunosorbent assay (ELISA) with kits from R&D Systems andBiolegend, respectively. GM-CSF and IL-12 secretion levels are reportedin units of ng/10⁶ cells/24 hours.

Results of Transgene Expression in the Individual Cell Lines afterAdaptation to Xeno-Free Media

DBTRG-05MG cells used for the adaptation process were modified to reduceTGFβ1 expression and to express CD40L and IL-12. Cells proliferatedstably when weaned to grow in 100% A1-XFR media over the course of 4-6weeks, but doubling times increased to 586.8 hours compared to 38.3hours of unmodified parental cells grown in FBS-containing media (Table123). Direct plating in A1-XFR media resulted in proliferation arrestand cell death. Analysis of modified DBTRG-05MG cells adapted to grow inA1-XFR media showed that CD40L is expressed and secretion of IL-12 isdetected and quantified to be 196.5 ng/10⁶/24 hrs, while secretion ofTGFβ1 is reduced by 88% from compared to unmodified parental DBTRG-05MGcells grown in FBS Unmodified DBTR-05MG cells do not express CD40L orproduce IL-12.

LN-229 cells used for the adaptation process were modified to reduceTGFβ1 expression and to overexpress CD40L, GM-CSF and IL-12. Cellsproliferated stably when directly plated in 100% A1-XFR media with adoubling time of 59.7 hours compared to 34.5 hours of unmodifiedparental cells grown in FBS-containing media (Table 123). When weaned togrow in 100% A2-XFR media over the course of 4-6 weeks, doubling timewas 71 hours (Table 4). Analysis of modified LN-229 cells adapted togrow in A1-XFR and A2-XFR media showed that CD40L is expressed andsecretion of IL-12 is detected and quantified to be 527 ng/10⁶/24 hrs(A1D) and 603 ng/10⁶/24 hrs (A2W), GM-CSF detected and quantified to be2029.8 ng/10⁶/24 hrs (A1D) and 2505.8 ng/10⁶/24 hrs (A2W) and TGFβ1levels were decreased by 79.2% (A1D) or 78.9% (A2W) compared tounmodified parental cells grown in FBS. Unmodified LN-229 cells do notexpress CD40L or produce IL-12 or GM-CSF.

GB1 cells used for the adaptation process were modified to havedecreased TGFβ1 expression and to overexpress CD40L and IL-12. Cellsproliferated stably when plated directly in 100% A1-XFR media with adoubling time of 144.1 hours compared to 37.9 hours of unmodifiedparental cells grown in FBS-containing media (Table 123). When weaned togrow in 100% A1-XFR media over the course of 4-6 weeks, the doublingtime was 597.2 hours, and 266.8 hours in A2-XFR media (Table 4).Analysis of modified GB1 cells adapted to grow in A1-XFR and A2-XFRmedia showed that CD40L is expressed and secretion of IL-12 is detectedand quantified to be 117.5 ng/10⁶/24 hrs (A1D), 76.6 ng/10⁶/24 hrs (A1W)and 72.0 ng/10⁶/24 hrs (A2W), and TGFβ1 levels were decreased by 64.3%(A1D), 74.6% (A1W) and 90.8% (A2W) compared to unmodified parental cellsgrown in FBS. Unmodified GB1 cells do not express CD40L or produceIL-12.

KNS-60 cells used for the adaptation process were modified to expressdecreased levels of TGFβ1 and TGFβ2, and to overexpress CD40L, GM-CSFand IL-12. Cells proliferated stably when weaned to grow in 100% A1-XFRmedia with a doubling time of 674.2 hours compared to 40.0 hours ofunmodified parental cells grown in FBS-containing media (Table 123).When weaned to grow in 100% A2-XFR media over the course of 4-6 weeks,the doubling time was 303.8 hours (Table 4). Analysis of modified KNS-60cells adapted to grow in A1-XFR and A2-XFR media showed that CD40L isexpressed and secretion of IL-12 is detected and quantified to be 700.0ng/10⁶/24 hrs (A1W) and 482.2 ng/10⁶/24 hrs (A2W), secretion of GM-CSFis detected and quantified to be 182.5 ng/10⁶/24 hrs (A1W) and 156.9ng/10⁶/24 hrs (A2W), and TGFβ1 levels were decreased by 83.2% (A1W) and87.7% (A2W) and TGFβ2 levels were decreased by 92.6% (A1W) and 94.7%(A2W) compared to unmodified parental cells grown in FBS. UnmodifiedKNS-60 cells do not express CD40L or produce IL-12 or GM-CSF.

SF-126 cells used for the adaptation process were modified to expressdecreased levels of TGFβ1 and TGFβ2, and to overexpress CD40L, GM-CSFand IL-12. Cells proliferated stably when weaned to grow in 100% A1-XFRmedia with a doubling time of 172.1 hours compared to 28.3 hours ofunmodified parental cells grown in FBS-containing media (Table 123).When weaned to grow in 100% A2-XFR media over the course of 4-6 weeks,the doubling time was 456.6 hours (Table 4). Analysis of modified SF-126cells adapted to grow in A1-XFR and A2-XFR media showed that CD40L isexpressed and secretion of IL-12 is detected and quantified to be 671.2ng/10⁶/24 hrs (A1W) and 684.9 ng/10⁶/24 hrs (A2W), secretion of GM-CSFis detected and quantified to be 51.2 ng/10⁶/24 hrs (A1W) and 39.3ng/10⁶/24 hrs (A2W), and TGFβ1 levels were decreased by 86.9% (A1W) and91.2% (A2W) and TGFβ2 levels were decreased by 80.4% (A1W) and 98.8%(A2W) compared to unmodified parental cells grown in FBS. UnmodifiedSF-126 cells do not express CD40L or produce IL-12 or GM-CSF.

In conclusion, all five modified GBM vaccine component cell lines stablyadapted to xeno-free media formulations. The cells proliferated at asteady rate, inserted transgene expression was maintained and thereduction of TGFβ1 and TGFβ2 was also retained.

TABLE 123 Doubling time of vaccine component cell lines inFBS-containing media and xeno-free media DT [hours] DT [hours] A1 mediaDT [hours] DT [hours] of parental Direct A1 media, A2 media, Cell linecell line (A1D) Wean (A1W) Wean (A2W) DBTRG-05MG 38.3 n/a 586.8 n/aLN-229 34.5 59.7 n/a 71 GB1 37.9 144.1 597.2 266.8 KNS-60 40 n/a 674.2303.8 SF-126 28.3 n/a 172.1 456.6 DT: doubling time; DT representsaverage values according to Conversion Reports

Example 38: Adaptation of NSCLC Vaccine Component Cell Lines to Growthin Xeno-Free Media

Overview of Adaptation Process

The six component cell lines (NCI-H23, A549, NCI-H460, DMS 53, LK-2 andNCI-H520) of the NSCLC vaccine composition were sequentially adapted togrowth in media that is xeno-free, serum-free and containing nonon-human elements. For each of the six cell lines, four xeno-free mediaformulations were tested. The media formulations are KSC pH 7.2, KSC pH6.8, KSR pH 7.2 and KSR pH 6.8. An additional control condition of cellsin regular culture media composed of RPMI supplemented with 10% FBS,L-Glutamine, sodium pyruvate and HEPES was also maintained. Eachxeno-free media formulation was composed of a different base medium (KSCor KSR) with 10% human serum albumin (HSA) as a xeno-free serumreplacement and antibiotics were added to the media to maintain theexpression of the inserted transgenes as shown in Table 124. As thetotal protein content of the xeno-free media was comparable to that ofmedia containing FBS, the antibiotic levels used for selection was thesame as in FBS-based media. Additionally, each media formulation wastested at two levels of oxygen- normal 21% oxygen and low 3% oxygen. Toconfirm adaptation to the xeno-free media formulations, the cells wereobserved for their ability to proliferate in the test media. Conditionsthat showed cell death based on visual observation of non-adherentfloaters that were non-viable upon staining with a viability dye wereterminated. The cells that had a morphology similar to the control FBSwells, were stably growing in XF media and were under antibioticselection for at least 3 weeks were harvested and the expression ofinserted transgenes analyzed.

TABLE 124 NSCLC antibiotic concentrations for selection of insertedtransgenes Cell Line Puro* Blast* Hygro* Neo* Zeo* NCI-H23 1 2 300 60050 A549 1 2 800 600 1200 NCI-H460 1 2 300 600 1200 DMS 53 n/a 4 200 600n/a LK-2 1 2 200 200 n/a NCI-H520 1 2 300 600 n/a *All selectionantibiotic concentrations are in μg/mL. n/a, selection antibiotic notused for cell line. Puro, Puromycin. Blast, Blasticidin. Hygro,Hygromycin. Neo, Neomycin (G418). Zeo, Zeocin.

Analysis of Transgene Expression in Cell Lines Grown in Xeno-Free Media

Each of the six vaccine component cell lines were screened for growth in4 media formulations and 2 oxygen levels. The conditions that showedstable cell growth, minimal cell death and morphology comparable to thecells grown in FBS were analyzed for expression of transgenes. Secretedcytokines were measured using an enzyme linked immunosorbent assay(ELISA). Briefly, for each sample, two-four dilutions of the supernatantwere run, and the data shown is the average of all conditions tested,normalized for dilution factor and cell count. TGFβ1 and TGFβ2 levelswere determined using an enzyme-linked immunosorbent assay (ELISA) (R&DSystems). TGFβ1 and TGFβ2 secretion is reported in units of pg/ml/10⁶cells. GM-CSF and IL-12 levels were determined using an enzyme-linkedimmunosorbent assay (ELISA) with kits from R&D Systems and Biolegendrespectively. GM-CSF and IL-12 secretion levels are reported in units ofng/ml/10⁶ cells. The expression of CD40L was assessed by flow cytometry.Briefly, after being harvested the cells were stained withphycoerythrin-conjugated anti-human CD40L (clone TRAP1). The labelledcells were analyzed by flow cytometry using a LSR Fortessa Flowcytometer.

Results of Transgene Expression in the Individual Cell Lines afterAdaptation to Xeno-Free Media

NCI-H23 cells showed stable growth in treatment medias 4 (KSR pH 7.2)and 5 (KSR pH 6.8) under normal 21% oxygen condition. The cells failedto proliferate in the other treatment conditions. The expression of thesurface protein CD40L was found to be stable and expressed at levelscomparable to the cells grown in FBS. Secretion of IL-12 and GM-CSF werefound to be increased in the xeno-free media formulations when comparedto FBS 1.7-fold (IL-12 media 4 and 5) and 2.3-fold (GM-CSF media 4 and5) respectively. The reduction of TGFβ1 and TGFβ2 was found to begreater in the XF media with the levels of TGFβ1 10-fold less in media 4and 7-fold less in media 5, while TGFβ2 was not detectable in the XFmedia, when compared to cells in FBS containing media.

A549 cells showed stable growth in treatment medias 4 (KSR pH 7.2) and 5(KSR pH 6.8) under normal 21% oxygen, and in treatment media 5 under low3% oxygen conditions. The cells failed to proliferate in the othertreatment conditions. The expression of the surface protein CD40L wasfound to be stable and expressed at levels comparable to the cells grownin FBS.

Secretion of IL-12 was found to be comparable to FBS in xeno-free media4 grown in normal 21% oxygen, increased by 1.6-fold in media 5 in normal21% oxygen condition and decreased by 0.6-fold in media 5 under low 3%oxygen condition. Secretion of GM-CSF was found to be comparable to FBSin xeno-free media 4 grown in normal 21% condition, increased by1.4-fold in media 5 in normal 21% oxygen condition and decreased by0.6-fold in media 5 under low 3% oxygen condition. The reduction ofTGFβ1 and TGFβ2 was found to be greater in the XF media with the levelsof TGFβ1 3.4-fold less in media 4 under normal 21% oxygen and 3.1-foldless in media 5 under normal 21% oxygen and 2-fold less in media 5 underlow 3% oxygen, while TGFβ2 was reduced by 2.2-fold in treatment media 4under normal 21% oxygen and was not detectable in the XF media 5 in low3% or normal 21% oxygen condition, when compared to cells in FBScontaining media.

NCI-H460 cells showed stable growth in treatment medias 4 (KSR pH 7.2)and 5 (KSR pH 6.8) under normal 21% oxygen condition. The cells failedto proliferate in the other treatment conditions. The expression of thesurface protein CD40L was found to be stable and expressed at levelscomparable to the cells grown in FBS. Secretion of IL-12 was found to beincreased in the xeno-free media formulations when compared to FBS,3.2-fold in media 4 and 1.7-fold in media 5. GM-CSF was also increasedin the xeno-free medias, 3.5-fold in media 4 and 1.8-fold in media 5.The reduction of TGFβ1 and TGFβ2 was found to be greater in the XF mediawith the levels of TGFβ1 not detectable in the XF medias 4 and 5 andTGFβ2 reduced 1.6-fold in media 4 and 3.4-fold in media 5, when comparedto cells in FBS containing media.

DMS 53 cells showed stable growth in treatment medias 4 (KSR pH 7.2) and5 (KSR pH 6.8) under normal 21% oxygen condition. The cells failed toproliferate in the other treatment conditions. The expression of thesurface protein CD40L was found to be stable and expressed at levelscomparable to the cells grown in FBS. GM-CSF secretion was increased inthe xeno-free medias, 3.5-fold in media 4 and 1.8-fold in media 5. TGFβ2levels was found to be greater in the XF media 4 by 1.2-fold anddecreased by 1.8-fold in media 5, when compared to cells in FBScontaining media. The cell line was not modified to overexpress IL-12 orhave a knock down in TGFβ1 levels.

LK-2 cells showed stable growth in treatment medias 4 (KSR pH 7.2) and 5(KSR pH 6.8) under normal 21% oxygen condition. The cells failed toproliferate in the other treatment conditions. The expression of thesurface protein CD40L was found to be stable and expressed at levelscomparable to the cells grown in FBS. GM-CSF secretion was increased inthe xeno-free medias, 2.8-fold in media 4 and 3.1-fold in media 5. TGFβ1levels were not detectable in xeno-free media, and TGFβ2 levels weredecreased by 6-fold in media 4 and 2.3-fold in media 5, when compared tocells in FBS containing media. The cell line was not modified tooverexpress IL-12.

NCI-H520 cells showed stable growth in treatment medias 4 (KSR pH 7.2)and 5 (KSR pH 6.8) under normal 21% oxygen and low 3% oxygen conditions.The cells failed to proliferate in the other treatment conditions. Theexpression of the surface protein CD40L was found to be stable andexpressed at levels comparable to the cells grown in FBS. Secretion ofGM-CSF was increased in xeno-free media 4 and 5 grown in normal 21%conditions by 1.5 and 1.3-fold respectively. GM-CSF secretion was alsoincreased in cells grown in low 3% oxygen conditions- 2.1-fold in media4 and 2.2-fold in media 5. Secretion of TGFβ1 was increased 10-fold inmedias 4 and 5 under normal 21% oxygen, and not detectable when thecells were grown in low 3% oxygen. Secretion of TGFβ2 was decreased3-fold and 1.2-fold in medias 4 and 5 under normal 21% oxygen, and notdetectable when the cells were grown in low 3% oxygen. The cell line wasnot modified to overexpress IL-12.

In conclusion, all six modified NSCLC vaccine component cell lines werestably adapted to growth in xeno-free media conditions. The cellsretained the reduction of TGFβ1 and TGFβ2 secretion and the secretion ofGM-CSF and IL-12 was found to be comparable to or increased in thexeno-free formulations when compared to the modified cells grown in FBS.Expression of the surface protein CD40L was detected at levels similarto cells grown in FBS across all conditions tested.

Example 39: Allogeneic Tumor Cell Vaccine Platform

This Example provides the compositions and methods for using variousallogeneic tumor cell vaccines for the treatment and/or prevention ofcancer and/or to stimulate an immune response. Given the teachingprovided herein, in some embodiments the following cell linecombinations and modifications are embraced by the present disclosure.Other embodiments (e.g., alternative cell lines and/or modifications asprovided herein) are also contemplated.

TABLE 125 Small cell lung cancer vaccine TGFβ1 TGFβ2 CD276 GM- IL- CellLine KD KD KO CD40L CSF 12 TAAs  1. DMS 114 x ND x x * * **  2. NCI-H196x x x x * * **  3. NCI-H1092 ND x x x * * **  4. SBC-5 x ND x x * * ** 5. NCI-H510A x x x x * * **  6. NCI-H889 x x x x * * **  7. NCI-H1341 xND x x * * **  8. NCIH-1876 x x x x * * **  9. NCI-H2029 ND x x x * * **10. NCI-H841 x ND x x * * ** 11. NCI-H1694 x ND x x * * ** DMS 53 ND x xx x x ND ND = not done * = One or more cell lines will be transduced insome embodiments to produce at least 15,000 ng per cocktail of GM-CSFand at least 4,000 ng of IL-12 per cocktail ** = Small cell lung cancervaccine will be modified in some embodiments to express one or more ofthe following TAAs: MAGEA1 and DLL3.

TABLE 126 Liver cancer vaccine TGFβ1 TGFβ2 CD276 GM- Cell Line KD KD KOCD40L CSF IL-12 TAAs 1. Hep-G2 x ND x x * * ** 2. JHH-2 x x x x * * **3. JHH-4 x x x x * * ** 4. JHH-6 x x x x * * ** 5. Li7 x x x x * * ** 6.HLF x x x x * * ** 7. HuH-6 x ND x x * * ** 8. JHH-5 x x x x * * ** 9.HuH-7 x x x x * * ** DMS 53 ND x x x x x ND ND = not done * = One ormore cell lines will be transduced in some embodiments to produce atleast 15,000 ng per cocktail of GM-CSF and at least 4,000 ng of IL-12per cocktail ** = Liver cancer vaccine will be modified in someembodiments to express one or more of the following TAAs: CEA (CEACAM5),MAGEA1, WT1, and PSMA (FOLH1).

TABLE 127 Kidney cancer vaccine TGFβ1 TGFβ2 CD276 GM- IL- Cell Line KDKD KO CD40L CSF 12 TAAs 1. A-498 x x x x * * ** 2. A-704 x x x x * * **3. 769-P x ND x x * * ** 4. 786-O x x x x * * ** 5. ACHN x x x x * * **6. KMRC-1 x x x x * * ** 7. KMRC-2 x x x x * * ** 8. VMRC-RCZ x x xx * * ** 9. VMRC-RCW x x x x * * ** DMS 53 ND x x x x x ND ND = notdone * = One or more cell lines will be transduced in some embodimentsto produce at least 15,000 ng per cocktail of GM-CSF and at least 4,000ng of IL-12 per cocktail ** = Kidney cancer vaccine will be modified insome embodiments to express one or more of the following TAAs: MAGEA1,DLL3, CEA (CEACAM5), and PSMA (FOLH1)

TABLE 128 Pancreatic cancer vaccine TGFβ1 TGFβ2 CD276 GM- Cell Line KDKD KO CD40L CSF IL-12 TAAs 1. PANC-1 x x x x * * ** 4. KP-3 x x x x * *** 5. KP-4 x ND x x * * ** 7. SUIT-2 x x x x * * ** 8. AsPC-1 x x xx * * ** 9. PSN1 x x x x * * ** DMS 53 ND x x x x x ND ND = not done * =One or more cell lines will be transduced in some embodiments to produceat least 15,000 ng per cocktail of GM-CSF and at least 4,000 ng of IL-12per cocktail ** = Pancreatic cancer vaccine will be modified in someembodiments to express one or more of the following TAAs: PSMA (FOLH1),BORIS (CTCFL), DLL3

TABLE 129 Esophageal cancer vaccine TGFβ1 TGFβ2 CD276 GM- Cell Line KDKD KO CD40L CSF IL-12 TAAs  1. TE-10 x x x x * * **  2. TE-6 x x x x * ***  3. TE-4 x x x x * * **  4. EC-GI-10 x x x x * * **  5. OE33 x x xx * * **  6. TE-9 x x x x * * **  7. TT x ND x x * * **  8. TE-11 x x xx * * **  9. OE19 x ND x x * * ** 10. OE21 x x x x * * ** DMS 53 ND x xx x x ND ND = not done * = One or more cell lines will be transduced insome embodiments to produce at least 15,000 ng per cocktail of GM-CSFand at least 4,000 ng of IL-12 per cocktail ** = Esophageal cancervaccine will be modified in some embodiments to express one or more ofthe following TAAs: WT1 and PSMA (FOLH1).

TABLE 130 Endometrial cancer vaccine TGFβ1 TGFβ2 CD276 GM- IL- Cell LineKD KD KO CD40L CSF 12 TAAs  1. SNG-M x ND x x * * **  2. HEC-1-B x ND xx * * **  3. JHUEM-3 x x x x * * **  4. RL95-2 ND ND x x * * **  5.MFE-280 x ND x x * * **  6. MFE-296 x x x x * * **  7. TEN x ND x x * ***  8. JHUEM-2 x ND x x * * **  9. AN3-CA ND x x x * * ** 10. Ishikawa xx x x * * ** DMS 53 ND x x x x x ND ND = not done * = One or more celllines will be transduced in some embodiments to produce at least 15,000ng per cocktail of GM-CSF and at least 4,000 ng of IL-12 per cocktail **= Endometrial cancer vaccine will be modified in some embodiments toexpress one or more of the following TAAs: BORIS (CTCFL), WT1, PSMA(FOLH1)

TABLE 131 Melanoma cancer vaccine TGFβ1 TGFβ2 CD276 GM- IL- Cell Line KDKD KO CD40L CSF 12 TAAs  1. RPMI-7951 x x x x * * **  2. MeWo x ND xx * * **  3. Hs 688(A).T x ND x x * * **  4. COLO 829 x ND x x * * ** 5. C32 x x x x * * **  6. A-375 x ND x x * * **  7. Hs 294T x x x x * ***  8. Hs 695T x ND x x * * **  9. Hs 852T x ND x x * * ** 10. A2058 xND x x * * ** DMS 53 ND x x x x x ND ND = not done * = One or more celllines will be transduced in some embodiments to produce at least 15,000ng per cocktail of GM-CSF and at least 4,000 ng of IL-12 per cocktail **= Melanoma cancer vaccine will be modified in some embodiments toexpress one or more of the following TAAs: MART-1 (MLANA), TYRP1, andPSMA (FOLH1)

TABLE 132 Mesothelioma cancer vaccine TGFβ1 TGFβ2 CD276 GM- Cell Line KDKD KO CD40L CSF IL-12 TAAs 1. NCI-H28 x ND x x * * ** 2. MSTO- x ND xx * * ** 211H 3. IST-Mes1 x x x x * * ** 4. ACC- x x x x * * ** MESO-15. NCI- x x x x * * ** H2052 6. NCI- x ND x x * * ** H2452 7. MPP 89 x xx x * * ** 8. IST-Mes2 x ND x x * * ** DMS 53 ND x x x x x ND ND = notdone * = One or more cell lines will be transduced in some embodimentsto produce at least 15,000 ng per cocktail of GM-CSF and at least 4,000ng of IL-12 per cocktail ** = Mesothelioma cancer vaccine will bemodified in some embodiments to express one or more of the followingTAAs: WT1, BORIS (CTCFL), and MAGEA1.

Example 40: Improving Breadth and Magnitude of Vaccine-Induced CellularImmune Responses by Introducing Non-Synonymous Mutations (NSM) intoPrioritized Full-Length Tumor Associated Antigens (TAAs)

Cancer immunotherapy through induction of anti-tumor cellular immunityhas become a promising approach targeting cancer. Many therapeuticcancer vaccine platforms are targeting tumor associated antigens (TAAs)that are overexpressed in tumor cells, however, a cancer vaccine usingthese antigens must be potent enough to break tolerance. The cancervaccines described in various embodiments herein are designed with thecapacity to elicit broad and robust cellular responses against tumors.Neoepitopes are non-self epitopes generated from somatic mutationsarising during tumor growth. Tumor types with higher mutational burdenare correlated with durable clinical benefit in response to checkpointinhibitor therapies. Targeting neoepitopes has many advantages becausethese neoepitopes are truly tumor specific and not subject to centraltolerance in the thymus. A cancer vaccine encoding full length TAAs withneoepitopes arising from nonsynonymous mutations (NSMs) has potential toelicit a more potent immune response with improved breadth andmagnitude.

Antigen Design Process

TAA Selection and Prioritization

TAAs are self-antigens that are either preferentially or abnormallyexpressed in tumors, but may be expressed at some level in normal cellsas well. As described herein, selecting and prioritizing TAAs as vaccinetargets is a critical step for cancer vaccine development. Multiplecriteria were utilized for TAA evaluation and selection. First, TAAswere identified and grouped into multiple categories including:

A. Proliferation

B. Adhesion, migration and metastasis

C. Angiogenesis

D. Cancer stem cell targets

E. Unknown function

Additionally, the tissue specificity of the TAAs in each group wasevaluated and the percentage of tumor samples with overexpression ofeach TAA was determined. Protein expression data measured by IHC arepreferred whenever it is applicable. Expression data from The HumanProtein Atlas were collected where no expression data is available.Lastly, TAAs in each group were prioritized and TAAs were selected basedon the criteria described. As an example, the GBM TAAs are summarized inTable 133 below after TAA selection and prioritization.

TABLE 133 GBM Prioritized TAAs TAA Group A: Cell proliferation IL-13RaSurvivin MAGE-1 hTERT WT1 Group B: Angiogenesis PSMA EphA2 Group C: CSCtargets Tenascin C (TNC) hTE.RT

Expression Profile for Component Tumor Cell Lines and TAA Identificationfor Design and Insertion

In order to determine whether the selected prioritized TAAs needed to beoverexpressed in the component cell lines that comprise the vaccinecompositions, expression profiles of all component cell lines for eachindication was created to determine whether the endogenous expression ofselected TAAs in these cell lines could be found. Expression of TAAs inthe potential component cell lines was determined using RNA-seq datadownloaded from the publicly available Cancer Cell Line Encyclopedia(CCLE) database (www.broadinstitute.org/ccle; Barretina, J et al.Nature. 2012) between Oct. 7, 2019-May 20, 2020. The HUGO GeneNomenclature Committee gene symbol was entered into the CCLE search andmRNA expression was downloaded for each TAA. The expression of a TAA wasconsidered positive if the RNA-seq value (FPKM) was greater than 0.Among the prioritized TAAs, those that were not expressed by any celllines or only expressed by one cell line comprising the therapeuticcombination of cell lines were identified for design and insertion. Anantigen could also be selected for design and insertion when it isexpressed by more than one cell line but its RNA expression level isabove 1.0 FPKM in only one cell line. An example of TAA expressionprofile (heat map) of various GBM cell lines is shown in FIG. 78.

The expression of prioritized TAAs listed in Table 66 in GBM cell lineswas determined using the data in FIG. 78. As indicated in FIG. 78, nocell lines exhibit positive hTERT or PSMA expression (the RNA-seq valuesfor hTERT and PSMA are all negative), while GB49 is the only cell linethat expresses MAGE-A1. As a result, design/enhancement andoverexpression of hTERT, PSMA and MAGE-A1 in selected GBM cell lines wasperformed.

Antigen Design Methods

After the TAAs that need to be overexpressed were selected, in order toincrease the breadth and magnitude of antigen-specific cellular immuneresponses, a multiphase design strategy was utilized to generatemodified TAAs with frequently occurring non-synonymous mutations incancer patients.

Patient tumor sample data were downloaded from the publicly availabledatabase cBioPortal (cbioportal.org) database (Cerami, E. et al. CancerDiscovery. 2012; Gao, J. et al. Sci Signal. 2013) between Feb. 23,2020-Jun. 2, 2020. The dataset of “curated set of nonredundant studies”was used and it contained 176 studies with whole exome or transcriptomesequencing of 46,706 tumor samples derived from 44,354 cancer patients.Table 134 lists the name, site of the primary tumor(s), number ofsamples, and the cBioPortal literature citation of the queried 176studies.

TABLE 134 Cancer Type/ Sample Study Name Primary Organ Site # cBioPortalStudy Citation Adenoid Cystic Carcinoma Project Adrenal Gland 1049Multi-Institute, 2019 Adrenocortical Carcinoma Adrenal Gland 92 TCGA,PanCancer Atlas Ampullary Carcinoma Ampulla of Vater 160 Baylor, CellReports 2016 Cholangiocarcinoma Biliary Tract 15 National Cancer Centerof Singapore, Nat Genet 2013 Cholangiocarcinoma Biliary Tract 8 NationalUniversity of Singapore, Nat Genet 2013 Cholangiocarcinoma Biliary Tract36 TCGA, PanCancer Atlas Intrahepatic Cholangiocarcinoma Biliary Tract40 JHU, Nat Genet 2013 Intrahepatic Cholangiocarcinoma Biliary Tract 103Shanghai, Nat Commun 2014 Gallbladder Carcinoma Biliary Tract 32Shanghai, Nat Genet 2014 Bladder Cancer Bladder/Urinary Tract 109 MSKCC,Eur Urol 2014 Bladder Cancer Bladder/Urinary Tract 97 MSKCC, J Clin Onco2013 Bladder Urothelial Carcinoma Bladder/Urinary Tract 99 BGI, NatGenet 2013 Bladder Urothelial Carcinoma Bladder/Urinary Tract 50DFCI/MSKCC Cancer Discov 2014 Bladder Urothelial CarcinomaBladder/Urinary Tract 411 TCGA, PanCancer Atlas Urothelial CarcinomaBladder/Urinary Tract 72 Cornell/Trento, Nat Genet 2016 Upper TractUrothelial Cancer Bladder/Urinary Tract 85 MSK, Eur Urol 2015 UpperTract Urothelial Carcinoma Bladder/Urinary Tract 47Cornell/Baylor/MDACC, Nat Commun 2019 Ewing Sarcoma Bone 112 InstituteCurie, Cancer Discov 2014 Pediatric Ewing Sarcoma Bone 107 DFCI, CancerDiscov 2014 Colorectal Adenocarcinoma Bowel 619 DFCI, Cell Reports 2016Colorectal Adenocarcinoma Bowel 74 Genentech, Nature 2012 ColorectalAdenocarcinoma Bowel 594 TCGA, PanCancer Atlas Colorectal AdenocarcinomaBowel 138 MSKCC, Genome Biol 2014 Triplets Colon Adenocarcinoma Bowel 29CaseCCC, PNAS 2015 Colon Cancer Bowel 110 CPTAC-2 Prospective, Cell 2019Breast Fibroepithelial Tumors Breast 22 Duke-MUS, Nat Genet 2015 BreastCancer Breast 2509 METRABRIC, Nature 2012 & Nat Commun 2016 BreastCancer Breast 70 MSKCC, 2019 Breast Invasive Carcinoma Breast 65 BritishColumbia, Nature 2012 Breast Invasive Carcinoma Breast 103 Broad, Nature2012 Breast Invasive Carcinoma Breast 100 Sanger, Nature 2012 BreastInvasive Carcinoma Breast 1084 TCGA, PanCancer Atlas Metastatic BreastCancer Breast 216 INSERM, PLoS Med 2016 Metastatic Breast Cancer ProjectBreast 237 MBCP Provisional Data Set, February 2020 Adenoid CysticCarcinoma Breast Breast 12 MSKCC, J Pathol 2015 Brain Lower Grade GliomaCNS/Brain 514 TCGA, PanCancer Atlas Glioma CNS/Brain 91 MSK, 2018 LowGrade Gliomas CNS/Brain 61 UCSF, Science 2014 Glioblastoma MultiformeCNS/Brain 592 TCGA, PanCancer Atlas Medulloblastoma CNS/Brain 92 Broad,Nature 2012 Medulloblastoma CNS/Brain 37 PCGP, Nature 2012Medulloblastoma CNS/Brain 46 Sickkids, Nature 2016 Cervical SquamousCell Cervix 297 TCGA, PanCancer Atlas Carcinoma Esophageal Squamous CellEsophagus/Stomach 88 ICGC, Nature 2014 Carcinoma Esophageal SquamousCell Esophagus/Stomach 139 UCLA, Nat Genet 2014 Carcinoma GastricAdenocarcinoma Esophagus/Stomach 78 TMUCIH, PNAS 2015 EsophagealAdenocarcinoma Esophagus/Stomach 151 DFCI, Nat Genet 2013 EsophagealAdenocarcinoma Esophagus/Stomach 182 TCGA, PanCancer Atlas StomachAdenocarcinoma Esophagus/Stomach 100 Pfizer and UHK, Nat Genet 2014Esophageal Adenocarcinoma Esophagus/Stomach 440 TCGA, PanCancer AtlasEsophageal Adenocarcinoma Esophagus/Stomach 30 U Tokyo, Nat Genet 2014Uveal Melanoma Eye 28 QIMR, Oncotarget 2016 Uveal Melanoma Eye 80 TCGA,PanCancer Atlas Head and Neck Squamous Cell Head and Neck 74 Broad,Science 2011 Carcinoma Head and Neck Squamous Cell Head and Neck 32Johns Hopkins, Science 2011 Carcinoma Head and Neck Squamous Cell Headand Neck 523 TCGA, PanCancer Atlas Carcinoma Oral Squamous CellCarcinoma Head and Neck 40 MD Anderson, Cancer Discov 2013Nasopharyngeal Carcinoma Head and Neck 56 Singapore, Nat Genet 2014Adenoid Cystic Carcinoma Head and Neck 28 FMI, Am J Surg Pathl 2014Adenoid Cystic Carcinoma Head and Neck 25 JHU, Cancer Prey Res 2016Adenoid Cystic Carcinoma Head and Neck 102 MDA, Clin Cancer Res 2015Adenoid Cystic Carcinoma Head and Neck 10 MGH, Nat Gen 2016 AdenoidCystic Carcinoma Head and Neck 60 MSKCC, Nat Genet 2013 Adenoid CysticCarcinoma Head and Neck 24 Sanger/MDA, JCI 2013 Clear Cell Renal CellCarcinoma Kidney 35 DFCI, Science 2019 Kidney Renal Clear Cell Kidney 98BGI, Nat Genet 2012 Carcinoma Kidney Renal Clear Cell Kidney 78 IRC, NatGenet 2014 Carcinoma Kidney Renal Clear Cell Kidney 512 TCGA, PanCancerAtlas Carcinoma Renal Clear Cell Carcinoma Kidney 106 U Tokyo, Nat Genet2013 Kidney Chromophobe Kidney 65 TCGA, PanCancer Atlas Kidney RenalPapillary Cell Kidney 283 TCGA, PanCancer Atlas Carcinoma RenalNon-Clear Cell Carcinoma Kidney 146 Genentech, Nat Genet 2014Unclassified Renal Cell Carcinoma Kidney 62 MSK, Nature 2016 PediatricRhabdoid Tumor Kidney 72 TARGET, 2018 Rhabdoid Cancer Kidney 40 BCGSC,Cancer Cell 2016 Pediatric Wilms' Tumor Kidney 657 TARGET, 2018Hepatocellular Adenoma Liver 46 INSERM, Cancer Cell 2014 HepatocellularCarcinomas Liver 243 INSERM, Nat Genet 2015 Liver Hepatocellular Adenomaand Liver 19 MSK, PLoS One 2018 Carcinomas Liver HepatocellularCarcinoma Liver 231 AMC, Hepatology 2014 Liver Hepatocellular CarcinomaLiver 27 RIKEN, Nat Genet 2012 Liver Hepatocellular Carcinoma Liver 372TCGA, PanCancer Atlas Thoracic PDX Lung 139 MSK, Provisional Small CellLung Cancer Lung 80 Johns Hopkins, Nat Genet 2012 Small Cell Lung CancerLung 110 U Cologne, Nature 2015 Small Cell Lung Cancer Lung 20Multi-Institute, Cancer Cell 2017 Non-Small Cell Lung Cancer Lung 75MSK, Cancer Cell 2018 Non-Small Cell Lung Cancer Lung 327 TRACERx, NEJM2017 Non-Small Cell Lung Cancer Lung 41 University of Turin, Lung Cancer2017 Lung Adenocarcinoma Lung 183 Broad, Cell 2012 Lung AdenocarcinomaLung 566 TCGA, PanCancer Atlas Lung Adenocarcinoma Lung 163 TSP, Nature2008 Lung Squamous Cell Carcinoma Lung 487 TCGA, PanCancer Atlas AcuteLymphoid Leukemia Lymphoid 73 St. Jude, Nat Genet, 2016 Pediatric AcuteLymphoid Lymphoid 1978 TARGET, 2018 Leukemia - Phase II ChronicLymphocytic Leukemia Lymphoid 160 Broad, Cell 2013 Chronic LymphocyticLeukemia Lymphoid 537 Broad, Nature 2015 Chronic Lymphocytic LeukemiaLymphoid 506 IUOPA, Nature 2015 Chronic Lymphocytic Leukemia Lymphoid105 ICGC, Nat Genet 2011 Cutaneous T cell Lymphoma Lymphoid 43 ColumbiaU, Nat Genet 2015 Diffuse Large B Cell Lymphoma Lymphoid 135 DFCI, NatMed 2018 Diffuse Large B Cell Lymphoma Lymphoid 1001 Duke, Cell 2017Diffuse Large B Cell Lymphoma Lymphoid 48 TCGA, PanCancer Atlas DiffuseLarge B Cell Lymphoma Lymphoid 53 BCGSC, Blood 2013 Mantel Cell LymphomaLymphoid 29 IDIBIPS, PNAS 2013 Multiple Myeloma Lymphoid 211 Broad,Cancer Cell 2014 Non-Hodgkin Lymphoma Lymphoid 14 BCGSC, Nature 2011Primary Central Nervous System Lymphoid 19 Mayo Clinic, Clin Cancer Res2015 Lymphoma Acute Myeloid Leukemia or Myeloid 136 WashU, 2016Myelodysplastic Syndromes Acute Myeloid Leukemia Myeloid 672 OHSU,Nature 2018 Acute Myeloid Leukemia Myeloid 200 TCGA, PanCancer AtlasPediatric Acute Myeloid Leukemia Myeloid 1025 TARGET, 2018 HistiocytosisCobimetinib Myeloid 52 MSK, 2019 Myelodysplasia Myeloid 29 UTokyo,Nature 2011 Myeloproliferative Neoplasms Myeloid 151 CIMR, NEJM 2013MSK-IMPACT Mixed Cancer Types 10,945 MSKCC, Nat Med. 2017 MSS MixedSolid Tumors Mixed Cancer Types 249 Broad/Dana-Farber, Nat Genet 2018Metastatic Solid Cancers Mixed Cancer Types 500 UMich, Nature. 2017Pediatric Pan-Cancer Mixed Cancer Types 961 DKFZ, Nature 2017 PediatricPan-cancer Mixed Cancer Types 103 Columbia U, Genome Med 2016 PediatricPreclinical Testing Mixed Cancer Types 261 Maris, 2019 ConsortiumSUMMIT-Neratinib Basket Study Mixed Cancer Types 141 Multi-Institute,Nature 2018 Ovarian Serous Ovarian/Fallopian 585 TCGA, PanCancer AtlasCystadenocarcinoma Small Cell Carcinoma of the Ovary Ovarian/Fallopian12 MSKCC, Nat Genet 2014 Acinar Cell Carcinoma of the Pancreas 23 JHU, JPathol 2014 Pancreas Cystic Tumor of the Pancreas Pancreas 32 JohnsHopkins, PNAS 2011 Pancreatic Adenocarcinoma Pancreas 456 QCMG, Nature2016 Pancreatic Adenocarcinoma Pancreas 184 TCGA, PanCancer AtlasPancreatic Cancer Pancreas 109 UTSW, Nat Commun 2015 Insulinoma Pancreas10 Shanghai, Nat Commun 2013 Pancreatic Neuroendocrine Pancreas 10 JohnsHopkins, Science 2011 Tumors Pancreatic Neuroendocrine Pancreas 98Multi-Institute, Nature 2017 Tumors Malignant Peripheral Nerve SheathPeripheral Nervous 15 MSKCC, Nat Genet 2014 Tumor NeuroblastomaPeripheral Nervous 87 AMC Amsterdam, Nature 2012 NeuroblastomaPeripheral Nervous 56 Broad, Nature 2015 Pediatric NeuroblastomaPeripheral Nervous 1089 TARGET, 2018 Mesothelioma Pleura 87 TCGA,PanCancer Atlas Pleural Mesothelioma Pleura 22 NYU, Cancer Res 2015Prostate Cancer Prostate 18 MSK, 2019 Metastatic Prostate Prostate 61MCTP, Nature 2012 Adenocarcinoma Metastatic Prostate Prostate 444SU2C/PCF Dream Team, PNAS 2019 Adenocarcinoma Neuroendocrine ProstateCancer Prostate 114 Multi-Institute, Nat Med 2016 ProstateAdenocarcinoma Prostate 112 Broad/Cornell, Nat 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The non-redundant data set was queried with the HUGO Gene NomenclatureCommittee gene symbol for the antigen of interest. Missense mutationsoccurring in the target antigen were downloaded and sorted by frequencyof occurrence. Missense mutations occurring in ≥2 patient samples wereidentified and evaluated for the potential to induce neoepitopes usingthe publicly available NetMHCpan 4.0 database(https://services.healthtech.dtu.dk/service.php?NetMHCpan-4.0) (Jurtz V,et al. J Immunol. 2017). The HLA supertypes included are HLA-A*01:01,HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*26:01, HLA-B*07:02,HLA-B*08:01, HLA-B*27:05, HLA-B*39:01, HLA-B*40:01, HLA-B*58:01, andHLA-B*15:01.

TABLE 135 Supertype Representative A01 HLA-A*01:01 A02 HLA-A*02:01 A03HLA-A*03:01 A24 HLA-A*24:02 A26 HLA-A*26:01 B07 HLA-B*07:02 B08HLA-B*08:01 B27 HLA-B*27:05 B39 HLA-B*39:01 B44 HLA-B*40:01 B58HLA-B*58:01 B62 HLA-B*15:01

The threshold for strong binder was set at the recommended threshold of0.5, which means any peptides with predicted % rank lower than 0.5 willbe annotated as strong binders. The threshold for weak binder was set atthe recommended 2.0, which means any peptides with predicted % ranklower than 2.0 but higher than 0.5 will be annotated as weak binders.

To determine whether introduction of a NSM occurring ≥2 patient samplesinto the human native antigen would create a new epitope or change aweak binder to strong binder, a list of HLA-A and HLA-Bsupertype-restricted 9-mer epitopes including both strong binders andweak binders was first generated using human native TAA protein sequence(List #1). Then, starting from 5′ end of the human native antigen, eachspecific NSM was introduced to the human native antigen by replacing thenative residue at the same position with the NSM. The resulting antigenwith the NSM was used to generate a new list of HLA-A and HLA-Bsupertype-restricted epitopes including both strong binders and weakbinders (List #2). By comparing List #2 with List #1, the numbers of newepitopes (strong binders and weak binders) and abrogated epitopes werecalculated. If introduction of one specific NSM resulted in more newepitopes, then this NSM would be included in the human native TAA. Ifintroduction of one specific NSM created the same number of new epitopesand abrogated epitopes, but it changed more weak binders to strongbinders, the decision would still be made to include this NSM in thehuman native TAA. If there were fewer than 9 amino acid residues betweentwo NSMs, then evaluation were performed for each individual NSM and thecombination of two NSMs as well. Once the evaluation was completed,sequence alignment was performed to determine the protein sequenceidentity between the human native TAA and human TAA with NSMs. If thesequence identity is below 90%, then only NSMs occurring in ≥2 patientsamples that either creates new epitopes or change weaker binders tostrong binders were included.

As an example, the PSMA with NSMs was designed using the methoddescribed above. FIG. 129 shows the sequence alignment between humannative PSMA (NCBI Gene ID: 2346) and the designed PSMA with NSMs(modPSMA; SEQ ID NO: 38). The NSMs (the residues that are differentbetween huPSMA and modPSMA) are highlighted in gray. The sequenceidentity between huPSMA and modPSMA is 96.4%.

The HLA-A and HLA-B supertype-restricted epitopes for huPSMA and themodPSMA are summarized in Table 136. 49 NSMs occurring ≥2 times wereidentified for PSMA 27 were included in the modPSMA antigen sequence.Compared to native PSMA, modPSMA contains an additional 41 neoepitopesdue to the introduction of NSMs.

TABLE 136 Epitopes in Native and Designed (mod) PSMA Native Designed HLASupertype SB WB Total SB WB Total A01 3 16 19 3 17 20 A02 6 11 17 6 1319 A03 6 9 15 7 12 19 A24 7 12 19 8 14 22 A26 8 28 36 10 24 34 B07 6 915 9 8 17 B08 4 17 21 4 25 29 B27 5 16 21 8 16 24 B39 8 16 24 9 26 35B44 5 12 17 7 13 20 B58 9 10 19 9 13 22 B62 10 15 25 11 17 28 TotalEpitopes 77 171 248 91 198 289

The HLA-A and HLA-B supertype-restricted epitopes for human WT1 (NCBIGene ID: 7490) and the modWT1 (SEQ ID NO: 81) are summarized in Table137. 46 NSMs occurring ≥2 times were identified for WT1 and 28 wereincluded in the modWT1 antigen sequence. When compared to native WT1,modWT1 contains an additional 33 more neoepitopes due to theintroduction of NSMs.

TABLE 137 Epitopes in Native and Designed (mod) WT1 Native Designed HLASupertype SB WB Total SB WB Total A01 1 7 8 2 9 11 A02 3 3 6 3 5 8 A03 44 8 4 6 10 A24 0 5 5 0 7 7 A26 7 5 12 8 8 16 B07 3 11 14 2 15 17 B08 0 66 0 8 8 B27 4 6 10 4 6 10 B39 6 15 21 6 17 23 B44 1 10 11 2 11 13 B58 26 8 4 11 15 B62 6 6 12 6 10 16 Total Epitopes 37 84 121 41 113 154

The HLA-A and HLA-B supertype-restricted epitopes for human FSHR (NCBIGene ID: 2492) and the modFSHR (SEQ ID NO: 95) are summarized in Table138. 70 NSMs occurring ≥2 times were identified for FSHR and 26 wereincluded in the modFSHR antigen sequence. When compared to native FSHR,modFSHR contains 47 more neoepitopes due to the introduction of NSMs.

TABLE 138 Epitopes in Native and Designed (mod) FSHR Native Designed HLASupertype SB WB Total SB WB Total A01 4 12 16 5 17 22 A02 12 24 36 14 2842 A03 7 7 14 8 8 16 A24 12 18 30 12 19 31 A26 10 15 25 10 21 31 B07 716 23 8 15 23 B08 7 28 35 9 27 36 B27 6 10 16 7 12 19 B39 17 23 40 19 3049 B44 3 13 16 4 15 19 B58 6 24 30 6 26 32 B62 13 14 27 15 20 35 TotalEpitopes 104 204 308 117 238 355

The HLA-A and HLA-B supertype-restricted epitopes for human TERT (NCBIGene ID: 7015) and the modTERT (SEQ ID NO: 36) are summarized in Table139. 75 NSMs occurring ≥2 times were identified for TERT and 43 wereincluded in the modTERT antigen sequence. When compared to native TERT,modTERT contains 47 more neoepitopes due to the introduction of NSMs.

TABLE 139 Epitopes in Native and Design (mod) TERT HLA Native DesignedSupertype SB WB Total SB WB Total A01 4 15 19 4 15 19 A02 12 25 37 16 2642 A03 9 18 27 9 19 28 A24 11 20 31 12 24 36 A26 9 21 30 10 24 34 B07 2148 69 18 47 65 B08 28 39 67 30 42 72 B27 23 39 62 27 35 62 B39 24 43 6722 59 81 B44 8 15 23 8 15 23 B58 10 16 26 10 18 28 B62 12 35 47 12 37 49Total Epitopes 171 334 505 178 361 539

The HLA-A and HLA-B supertype-restricted epitopes for BORIS (NCBI GeneID: 140690) and the modBORIS (SEQ ID NO: 60) are summarized in Table140. 51 NSMs occurring ≥2 times were identified for BORIS and 33 wereincluded in the modBORIS antigen sequence. When compared to nativeBORIS, modBORIS contains 27 more neoepitopes due to the introduction ofNSMs.

TABLE 140 Epitopes in Native and Designed (mod) BORIS (CTCFL) HLA NativeDesigned Supertype SB WB Total SB WB Total A01 2 10 12 2 11 13 A02 0 5 51 4 5 A03 4 12 16 6 16 22 A24 2 4 6 5 3 8 A26 3 10 13 5 15 20 B07 1 1011 2 9 11 B08 6 14 20 8 14 22 B27 1 10 11 1 13 14 B39 10 15 25 9 18 27B44 9 16 25 6 18 24 B58 1 10 11 2 11 13 B62 4 13 17 4 16 20 TotalEpitopes 43 129 172 51 148 199

The HLA-A and HLA-B supertype-restricted epitopes for MSLN (NCBI GeneID: 10232) and the modMSLN (SEQ ID NO: 62) are summarized in Table 141.23 NSMs occurring ≥2 times were identified for MSLN and 13 were includedin the modMSLN antigen sequence. When compared to native MSLN, modMSLNcontains 23 more neoepitopes due to the introduction of NSMs.

TABLE 141 Epitopes in Native and Designed (mod) MSLN HLA Native DesignedSupertype SB WB Total SB WB Total A01 3 10 13 3 12 15 A02 5 12 17 6 1218 A03 4 6 10 4 6 10 A24 4 8 12 6 11 17 A26 11 14 25 11 14 25 B07 11 1627 9 19 28 B08 5 13 18 6 13 19 B27 2 6 8 2 8 10 B39 12 18 30 12 20 32B44 4 12 16 6 14 20 B58 3 7 10 3 12 15 B62 4 14 18 4 14 18 TotalEpitopes 68 136 204 72 155 227

The HLA-A and HLA-B supertype-restricted epitopes for TBXT (NCBI GeneID: 6862) and the modTBXT (SEQ ID NO: 79) are summarized in Table 142.44 NSMs occurring ≥2 times were identified for TBXT and 16 were includedin the modTBXT antigen sequence. When compared to native TBXT, modTBXTcontains 34 more neoepitopes due to the introduction of NSMs.

TABLE 142 Epitopes in Native and Designed (mod) TBXT HLA Native DesignedSupertype SB WB Total SB WB Total A01 6 4 10 8 5 13 A02 4 5 9 4 9 13 A032 4 6 3 6 9 A24 5 6 11 5 7 12 A26 2 5 7 2 8 10 B07 5 14 19 4 12 16 B08 46 10 5 9 14 B27 5 2 7 6 3 9 B39 6 18 24 9 23 32 B44 3 7 10 4 6 10 B58 62 8 7 4 11 B62 4 12 16 8 14 22 Total Epitopes 52 85 137 65 106 171

The HLA-A and HLA-B supertype-restricted epitopes for PRAME (NCBI GeneID: 23532) and the modPRAME (SEQ ID NO: 99) are summarized in Table 143.27 NSMs occurring ≥2 times were identified for PRAME and 20 wereincluded in the modPRAME antigen sequence. When compared to nativePRAME, modPRAME contains 35 more neoepitopes due to the introduction ofNSMs.

TABLE 143 Epitopes in Native and Designed (mod) FRAME HLA NativeDesigned Supertype SB WB Total SB WB Total A01 5 14 19 5 14 19 A02 16 2036 17 25 42 A03 5 14 19 5 14 19 A24 5 18 23 7 22 29 A26 2 20 22 3 23 26B07 9 17 26 9 18 27 B08 13 33 46 17 37 54 B27 4 10 14 3 10 13 B39 13 2336 16 24 40 B44 7 15 22 7 16 23 B58 2 19 21 1 21 22 B62 8 22 30 12 23 35Total Epitopes 89 225 314 102 247 349

The HLA-A and HLA-B supertype-restricted epitopes for TDGF1 (NCBI GeneID: 6997) and the modTDGF1 (SEQ ID NO: 89) are summarized in Table 144.9 NSMs occurring ≥2 times were identified for TDGF1 and 7 were includedin the modTDGF1 antigen sequence. When compared to native TDGF1,modTDGF1 contains 11 more neoepitopes due to the introduction of NSMs.

TABLE 144 Epitopes in Native and Designed (mod) TDGF1 HLA NativeDesigned Supertype SB WB Total SB WB Total A01 0 1 1 0 1 1 A02 2 5 7 2 46 A03 1 1 2 1 1 2 A24 1 5 6 1 5 6 A26 0 7 7 2 6 8 B07 5 6 11 6 8 14 B082 11 13 3 10 13 B27 1 3 4 2 5 7 B39 2 4 6 2 7 9 B44 1 1 2 2 1 3 B58 2 68 2 7 9 B62 2 4 6 2 4 6 Total Epitopes 19 54 73 25 59 84

The HLA-A and HLA-B supertype-restricted epitopes for FOLR1 (FBP) (NCBIGene ID: 2348) and the modFOLR1 (SEQ ID NO: 93) are summarized in Table145. 15 NSMs occurring ≥2 times were identified for FOLR1 and 9 wereincluded in the modFOLR1 antigen sequence. When compared to nativeFOLR1, modFOLR1 contains 7 more neoepitopes due to the introduction ofNSMs.

TABLE 145 Epitopes in Native and Designed (mod) FOLR1 HLA NativeDesigned Supertype SB WB Total SB WB Total A01 1 4 5 0 5 5 A02 3 8 11 48 12 A03 2 2 4 3 4 7 A24 2 6 8 2 8 10 A26 1 4 5 1 5 6 B07 5 5 10 5 3 8B08 5 5 10 4 4 8 B27 1 2 3 2 1 3 B39 5 6 11 5 8 13 B44 1 3 4 1 3 4 B58 79 16 7 11 18 B62 2 5 7 2 5 7 Total Epitopes 35 59 94 36 65 101

The HLA-A and HLA-B supertype-restricted epitopes for CLDN18 (NCBI GeneID: 51208) and the modCLDN18 (SEQ ID NO: 110) are summarized in Table146. 22 NSMs occurring ≥2 times were identified for CLDN18 and 11 wereincluded in the modCLDN18 antigen sequence. When compared to nativeCLDN18, modCLDN18 contains 22 more neoepitopes due to the introductionof NSMs.

TABLE 146 Epitopes in Native and Designed (mod) CLDN18 HLA NativeDesigned Supertype SB WB Total SB WB Total A01 5 3 8 5 3 8 A02 7 10 17 617 23 A03 3 6 9 4 4 8 A24 3 8 11 3 10 13 A26 9 13 22 9 17 26 B07 0 1 1 01 1 B08 0 3 3 0 5 5 B27 2 0 2 1 0 1 B39 2 6 8 2 8 10 B44 2 2 4 2 5 7 B587 6 13 6 10 16 B62 5 11 16 4 14 18 Total Epitopes 45 69 114 42 94 136

The HLA-A and HLA-B supertype-restricted epitopes for Ly6K (NCBI GeneID: 54742) and the modLy6K (SEQ ID NO: 112) are summarized in Table 147.9 NSMs occurring ≥2 times were identified for Ly6K and 7 were includedin the modLy6K antigen sequence. When compared to native Ly6K, modLy6Kcontains 6 more neoepitopes due to the introduction of NSMs.

TABLE 147 Epitopes in Native and Designed (mod) Ly6K HLA Native DesignedSupertype SB WB Total SB WB Total A01 1 4 5 0 6 6 A02 6 3 9 6 2 8 A03 02 2 0 2 2 A24 0 5 5 1 7 8 A26 0 7 7 0 6 6 B07 2 2 4 3 1 4 B08 1 7 8 2 79 B27 2 1 3 2 1 3 B39 0 2 2 0 2 2 B44 1 2 3 1 1 2 B58 2 4 6 2 6 8 B62 18 9 1 10 11 Total Epitopes 16 47 63 18 51 69

The HLA-A and HLA-B supertype-restricted epitopes for MAGEA10 (NCBI GeneID: 4109) and the modMAGEA10 (SEQ ID NO: 97) are summarized in Table148. 38 NSMs occurring ≥2 times were identified for MAGEA10 and 13 wereincluded in the modMAGEA10 antigen sequence. When compared to nativeMAGEA10, modMAGEA10 contains 29 more neoepitopes due to the introductionof NSMs.

TABLE 148 Epitopes in Native and Designed (mod) MAGEA10 HLA NativeDesigned Supertype SB WB Total SB WB Total A01 7 13 20 7 15 22 A02 2 810 4 8 12 A03 2 6 8 2 9 11 A24 0 4 4 2 4 6 A26 4 12 16 4 15 19 B07 2 7 92 8 10 B08 2 3 5 2 5 7 B27 1 3 4 1 3 4 B39 4 12 16 5 17 22 B44 5 6 11 59 14 B58 0 13 13 2 14 16 B62 3 9 12 5 9 14 Total Epitopes 32 96 128 41116 157

The HLA-A and HLA-B supertype-restricted epitopes for MAGEC2 (NCBI GeneID: 51438) and the modMAGEC2 (SEQ ID NO:87) are summarized in Table 149.45 NSMs occurring ≥2 times were identified for MAGEC2 and 8 wereincluded in the modMAGEC2 antigen sequence. When compared to nativeMAGEC2, modMAGEC2 contains 14 more neoepitopes due to the introductionof NSMs.

TABLE 149 Epitopes in Native and Designed (mod) MAGEC2 HLA NativeDesigned Supertype SB WB Total SB WB Total A01 4 14 18 6 13 19 A02 9 1019 8 11 19 A03 3 3 6 4 5 9 A24 5 13 18 5 14 19 A26 10 19 29 10 21 31 B075 15 20 5 14 19 B08 4 10 14 5 12 17 B27 2 0 2 3 1 4 B39 5 11 16 7 10 17B44 7 13 20 7 12 19 B58 5 17 22 7 18 25 B62 8 12 20 8 12 20 TotalEpitopes 67 137 204 75 143 218

The HLA-A and HLA-B supertype-restricted epitopes for FAP (NCBI Gene ID:2191) and the modFAP (SEQ ID NO:115) are summarized in Table 150. 59NSMs occurring ≥2 times were identified for FAP and 25 were included inthe modFAP antigen sequence. When compared to native FAP, modFAPcontains 22 more neoepitopes due to the introduction of NSMs.

TABLE 150 Epitopes in Native and Designed (mod) FAP HLA Native DesignedSupertype SB WB Total SB WB Total A01 15 38 53 14 40 54 A02 11 14 25 1613 29 A03 11 18 29 14 17 31 A24 12 36 48 15 36 51 A26 24 35 59 27 37 64B07 7 13 20 7 11 18 B08 14 21 35 16 20 36 B27 9 13 22 9 12 21 B39 9 3443 8 36 44 B44 6 15 21 6 15 21 B58 12 32 44 13 34 47 B62 17 28 45 17 3350 Total Epitopes 147 297 444 162 304 466

The HLA-A and HLA-B supertype-restricted epitopes for MAGEA1 (NCBI GeneID: 4100) and the modMAGEA1 (SEQ ID NO: 73) are summarized in Table 151.16 NSMs occurring ≥2 times were identified for MAGEA1 and 10 wereincluded in the modMAGEA1 antigen sequence. When compared to nativeMAGEA1, modMAGEA1 contains 7 more neoepitopes due to the introduction ofNSMs.

TABLE 151 Epitopes in Native and Designed (mod) MAGEA1 HLA NativeDesigned Supertype SB WB Total SB WB Total A01 5 7 12 6 6 12 A02 3 8 115 7 12 A03 2 3 5 2 4 6 A24 3 3 6 4 4 8 A26 2 13 15 3 16 19 B07 3 4 7 2 24 B08 3 4 7 3 3 6 B27 2 3 5 1 3 4 B39 2 8 10 2 10 12 B44 4 7 11 4 6 10B58 2 11 13 2 12 14 B62 1 6 7 2 7 9 Total Epitopes 32 77 109 36 80 116

TABLE 152 Native Sequences for Designed (mod) Antigens TAA Name NCBIGene Symbol (Gene ID) TERT TERT (7015) PSMA (FOLH1) FOLH1 (2346) MAGE A1MAGEA1 (4100) TBXT TBXT (6862) BORIS CTCFL (140690) FSHR FSHR (2492)MAGEA10 MAGEA10 (4109) MAGEC2 MAGEC2 (51438) WT1 WT1 (7490) FBP FOLR1(2348) TDGF1 TDGF1 (6997) Claudin 18 CLDN18 (51208) LY6K LY6K (54742)Mesothelin MSLN (10232) FAP FAP (2191) FRAME FRAME (23532)

The following table describes predicted epitopes for HLA-A and HLA-Bsupertypes for an exemplary combination of TAAs in GBM. Predictedepitopes for PSMA (SEQ ID NO: 70), modPSMA (SEQ ID NO: 38), native TERT(Gene ID 7015), modTERT (SEQ ID NO: 36), native MAGEA1 (Gene ID 4100),and modMAGEA1 (SEQ ID NO: 73) are indicated by HLA-A and HLA-Bsupertype. Table 153 demonstrates the combination of designed antigenscreates a total of 82 neoepitopes: modPSMA creates 41 neoepitopes,modTERT 34 neoepitopes, and modMAGEA1 7 neoepitopes. FIG. 130A shows thefrequency of HLA-A and HLA-B supertype pairs in a subset of 28,034high-resolution HLA allele and haplotype frequency data available fromdonors in the National Marrow Donor Program databases from four majorU.S. census categories of race and ethnicity (L Maiers, M., et al.(2007)). The HLA-A and HLA-B supertypes were assigned to HLA-A and HLA-Ballele pairs occurring in the top 25th percentile of HLA-A and HLA-Bhaplotype pairs for each ethnic subgroup (FIG. 130B) according to Lundtet al. (2004). If either the HLA A or B haplotype was not classifiedinto a supertype it was not included in the analysis (Outlier). Data forHLA-A and HLA-B pairs was downloaded from the publicly availabledatabasehttps://bioinformatics.bethematchclinical.org/hla-resources/haplotype-frequencies/high-resolution-hla-alleles-and-haplotypes-in-the-us-population/ on Mar. 15, 2020. If an HLA-A or HLA-B allele in the dataset did not fall into an HLA-A and HLA-B supertype according to Lundt etal. it was excluded from the data subset (FIG. 130C).

TABLE 153 HLA PSMA TERT MAGE A1 Supertype Native Designed NativeDesigned Native Designed A01 19 20 19 19 12 12 A02 17 19 37 42 11 12 A0315 19 27 28 5 6 A24 19 22 31 36 6 8 A26 36 34 30 34 15 19 B07 15 17 6965 7 4 B08 21 29 67 72 7 6 B27 21 24 62 62 5 4 B39 24 35 67 81 10 12 B4417 20 23 23 11 10 B58 19 22 26 28 13 14 B62 25 28 47 49 7 9 Total 248289 505 539 109 116 Epitopes

In one exemplary embodiment, neoepitopes existing in the cell lines of avaccine composition and induced by design in GBM are provided in Table154.

TABLE 154 Super Mod Mod Mod Design Existing type PSMA TERT MAGE A1 LN229A172 YKG1 KNS60 SF126 DMS53 Total Total Total A01 2 0 1 1 0 0 0 1 0 3 25 A02 2 5 1 0 0 1 1 0 0 8 2 10 A03 4 3 2 0 0 0 2 0 0 9 2 11 A24 3 5 2 10 1 0 0 2 10 4 14 A26 2 5 3 0 0 0 0 0 0 10 0 10 B07 2 2 2 0 1 0 0 1 2 64 10 B08 9 6 0 0 0 0 1 1 0 15 2 17 B27 4 3 0 0 0 0 0 0 0 7 0 7 B39 12 164 0 0 1 0 0 0 32 1 33 B44 3 1 0 0 0 0 0 0 0 4 0 4 B58 3 2 0 1 0 1 0 0 05 2 7 B62 3 3 1 1 0 0 1 0 0 7 2 9 Total 49 51 16 4 1 4 5 3 4 116 21 137

FIG. 131 shows the number of neoepitopes existing in the cell lines of avaccine composition and created by design in GBM recognized by donorsexpressing HLA-A and HLA-B supertype pairs within the population subsetsdescribed in Example 29 herein. The median number of neoepitopesrecognized in the four ethnic subpopulations is twenty.

FIG. 132 depicts the number of neoepitopes targeted by four differentmRNA immunotherapies. One mRNA immunotherapy targets a total of 34neoepitopes and the other three mRNA therapies target a total of 20neoepitopes. Humans express two pairs of HLA-A and HLA-B allelles. Thenumber of neoepitopes that an exemplary patient expressing the HLA-A andHLA-B allellic pairs in the HLA-A3 HLA-B7 and HLA-A1 HLA-B-8 supertypeswould recognize of the neoepitopes existing in the cell lines of avaccine composition and created by design in GBM is forty-three. Thenumber of epitopes recognized in twenty prioritized TAAs existing in avaccine composition GBM by all HLA-A and HLA-B supertype pairs rangesfrom 1,500 to 2,000.

Example 41: Clinical Protocol

An exemplary clinical protocol is provided in the following Example.

Dosage form: The vaccine composition is provided to a clinical site in apackage containing six vials, each vial comprising a therapeuticallyeffective amount of cells from a cancer cell line, as described inembodiments disclosed herein (thus six cell lines total). Three of thecell lines constitute Cocktail A and the other three cell linesconstitute Cocktail B, thus resulting in three Cocktail A vials, andthree Cocktail B vials. At the time of administration, the vials areremoved from the freezeer and thawed at room temperature for about 5 toabout 15 minutes. The contents of two of the Cocktail A vials areremoved by needle and syringe and are injected into the third Cocktail Avial. Similarly, the contents of two of the Cocktail B vials are removedby needle and syringe and injected into the third Cocktail B vial.

Route of Administration: After mixing, 0.3 mL Cocktail A is drawn into asyringe and administered as an intradermal injection in the upper arm.Similarly and concurrently, 0.3 mL Cocktail B is drawn into a syringeand administered as an intradermal injection in the thigh. The doseadministered is about 8×10⁶ (or optionally 1×10⁷) of each cell line fora total dose of about 2.4×10⁷ (or optionally 3×10⁷) cells at eachinjection site. Multiple doses are administered, and administration isalternated between the left and right arms and left and right thighs. Asdescribed herein, the 0.3 mL injection volume can be split into 3×0.1 mLor 2×0.15 mL.

In one embodiment, Cocktail A and Cocktail B comprises the modified celllines as set out in Table 45, 56, 65, 74, 83, 92, 101, 110, 119.According to some embodiments, the clinical protocol may be used forother indications and using other cocktails of cell line combinations,as described herein.

Dosing Regimen: In various embodiments, three cohorts will receiveadministration of the vaccine in combination with a checkpoint inhibitor(CPI) such as pembrolizumab. In these cohorts, the vaccine will beadministered in 21-day cycles to match administration of the CPI. Thefirst four doses will be administered every 21 days (up to day 63) andthen every 42 days for three additional doses (up to day 189). Patientswho continue to benefit from treatment will be allowed to continue toreceive the vaccine in combination with a CPI for five additional dosesat 42-day intervals (up to day 399) and then at 84-day intervals.

In a fourth cohort, the vaccine will be administered in combination withdurvalumab. The vaccine will be administered in either 14-day, 21-day or28-day cycles to match administration of durvalumab. For example, thefirst three doses will be administered every 14 days (up to day 28) andthen every 42 days for four additional doses (up to day 196). Patientswho continue to benefit from treatment will be allowed to continue toreceive the vaccine in combination with durvalumab for five additionaldoses at 42-day intervals (up to day 406) and then at 84-day intervals.As another example, the first three doses will be administered every 28days.

All patients will receive an oral dose of 50 mg/day (or 100 mg/day)cyclophosphamide for seven days prior to each administration of theinvestigational product.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made without departing fromthe spirit and scope of the disclosure. Accordingly, other embodimentsare within the scope of the following claims.

The invention claimed is:
 1. A unit dose comprising a first compositioncomprising cancer cell lines NCI-H460 (ATCC HTB-177), NCI-H520 (ATCCHTB-182), and A549 (ATCC CCL-185), and a second composition comprisingcancer cell lines DMS 53 (ATCC CRL-2062), LK-2 (JCRB0829), and NCI-H23(ATCC CRL-5800), wherein: (a) the NCI-H460 cell line is modified invitro to: 1) knockout expression of CD276 using a zinc-finger nucleasetargeting CD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ ID NO: 8),TGFI2 shRNA (SEQ ID NO: 24), IL-12 (SEQ ID NO: 10), membrane bound CD40L(SEQ ID NO: 3), TGFI1 shRNA (SEQ ID NO: 25), and modBORIS (SEQ ID NO:60); (b) the NCI-H520 cell line is modified in vitro to: 1) knockoutexpression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ IDNO: 26); and 2) express GM-CSF (SEQ ID NO: 8), membrane bound CD40L (SEQID NO: 3), TGFI1 shRNA (SEQ ID NO: 25), and TGFI2 shRNA (SEQ ID NO: 24);(c) the A549 cell line is modified in vitro to: 1) knockout expressionof CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 26);and 2) express GM-CSF (SEQ ID NO: 8), TGFI2 shRNA (SEQ ID NO: 24), IL-12(SEQ ID NO: 10), membrane bound CD40L (SEQ ID NO: 3), TGFI1 shRNA (SEQID NO: 25), modTBXT (SEQ ID NO: 79), modWT1 (SEQ ID NO: 81), and KRASdriver mutations G12D and G12V; (d) the DMS 53 cell line is modified invitro to: 1) knockout expression of CD276 using a zinc-finger nucleasetargeting CD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ ID NO: 8),TGFI2 shRNA (SEQ ID NO: 24), IL-12 (SEQ ID NO: 10), membrane bound CD40L(SEQ ID NO: 3), and TGFI1 shRNA (SEQ ID NO: 25); (e) the LK-2 cell lineis modified in vitro to: 1) knockout expression of CD276 using azinc-finger nuclease targeting CD276 (SEQ ID NO: 26); and 2) expressGM-CSF (SEQ ID NO: 8), membrane bound CD40L (SEQ ID NO: 3), TGFI1 shRNA(SEQ ID NO: 25), and TGFI2 shRNA (SEQ ID NO: 24); and (f) the NCI-H23cell line is modified in vitro to: 1) knockout expression of CD276 usinga zinc-finger nuclease targeting CD276 (SEQ ID NO: 26); and 2) expressGM-CSF (SEQ ID NO: 8), TGFI2 shRNA (SEQ ID NO: 24), IL-12 (SEQ ID NO:10), membrane bound CD40L (SEQ ID NO: 3), TGFI1 shRNA (SEQ ID NO: 25),and modMSLN (SEQ ID NO: 62).
 2. The unit dose of claim 1, wherein thefirst composition and second composition each comprise approximately1.0×10⁶-6.0×10⁷ cells of each cell line.
 3. The unit dose of claim 1,wherein the cell lines of (a)-(f) are irradiated.
 4. A compositioncomprising cancer cell lines NCI-H460 (ATCC HTB-177), NCI-H520 (ATCCHTB-182), and A549 (ATCC CCL-185), wherein: (a) the NCI-H460 cell lineis modified in vitro to: 1) knockout expression of CD276 using azinc-finger nuclease targeting CD276 (SEQ ID NO: 26); and 2) expressGM-CSF (SEQ ID NO: 8), TGFI2 shRNA (SEQ ID NO: 24), IL-12 (SEQ ID NO:10), membrane bound CD40L (SEQ ID NO: 3), TGFI1 shRNA (SEQ ID NO: 25),and modBORIS (SEQ ID NO: 60); (b) the NCI-H520 cell line is modified invitro to: 1) knockout expression of CD276 using a zinc-finger nucleasetargeting CD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ ID NO: 8),membrane bound CD40L (SEQ ID NO: 3), TGFI1 shRNA (SEQ ID NO: 25), andTGFI2 shRNA (SEQ ID NO: 24); and (c) the A549 cell line is modified invitro to: 1) knockout expression of CD276 using a zinc-finger nucleasetargeting CD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ ID NO: 8),TGFI2 shRNA (SEQ ID NO: 24), IL-12 (SEQ ID NO: 10), membrane bound CD40L(SEQ ID NO: 3), TGFI1 shRNA (SEQ ID NO: 25), modTBXT (SEQ ID NO: 79),modWT1 (SEQ ID NO: 81), and KRAS driver mutations G12D and G12V.
 5. Acomposition comprising cancer cell lines DMS 53 (ATCC CRL-2062), LK-2(JCRB0829), and NCI-H23 (ATCC CRL-5800), wherein: (a) the DMS 53 cellline is modified in vitro to: 1) knockout expression of CD276 using azinc-finger nuclease targeting CD276 (SEQ ID NO: 26); and 2) expressGM-CSF (SEQ ID NO: 8), TGFI2 shRNA (SEQ ID NO: 24), IL-12 (SEQ ID NO:10), membrane bound CD40L (SEQ ID NO: 3), and TGFI1 shRNA (SEQ ID NO:25); (b) the LK-2 cell line is modified in vitro to: 1) knockoutexpression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ IDNO: 26); and 2) express GM-CSF (SEQ ID NO: 8), membrane bound CD40L (SEQID NO: 3), TGFI1 shRNA (SEQ ID NO: 25), and TGFI2 shRNA (SEQ ID NO: 24);and (c) the NCI-H23 cell line is modified in vitro to: 1) knockoutexpression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ IDNO: 26); and 2) express GM-CSF (SEQ ID NO: 8), TGFI2 shRNA (SEQ ID NO:24), IL-12 (SEQ ID NO: 10), membrane bound CD40L (SEQ ID NO: 3), TGFI1shRNA (SEQ ID NO: 25), and modMSLN (SEQ ID NO: 62).
 6. The compositionof claim 4, wherein the composition comprises approximately 1.0×10⁷cells for each cell line.
 7. The composition of claim 5, wherein thecomposition comprises approximately 1.0×10⁷ cells for each cell line. 8.A kit comprising six vials, wherein each vial individually comprisescells of cancer cell lines NCI-H460 (ATCC HTB-177), NCI-H520 (ATCCHTB-182), A549 (ATCC CCL-185), DMS 53 (ATCC CRL-2062), LK-2 (JCRB0829),and NCI-H23 (ATCC CRL-5800), wherein: (a) the NCI-H460 cell line ismodified in vitro to: 1) knockout expression of CD276 using azinc-finger nuclease targeting CD276 (SEQ ID NO: 26); and 2) expressGM-CSF (SEQ ID NO: 8), TGFI2 shRNA (SEQ ID NO: 24), IL-12 (SEQ ID NO:10), membrane bound CD40L (SEQ ID NO: 3), TGFI1 shRNA (SEQ ID NO: 25),and modBORIS (SEQ ID NO: 60); (b) the NCI-H520 cell line is modified invitro to: 1) knockout expression of CD276 using a zinc-finger nucleasetargeting CD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ ID NO: 8),membrane bound CD40L (SEQ ID NO: 3), TGFI1 shRNA (SEQ ID NO: 25), andTGFI2 shRNA (SEQ ID NO: 24); (c) the A549 cell line is modified in vitroto: 1) knockout expression of CD276 using a zinc-finger nucleasetargeting CD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ ID NO: 8),TGFI2 shRNA (SEQ ID NO: 24), IL-12 (SEQ ID NO: 10), membrane bound CD40L(SEQ ID NO: 3), TGFI1 shRNA (SEQ ID NO: 25), modTBXT (SEQ ID NO: 79),modWT1 (SEQ ID NO: 81), and KRAS driver mutations G12D and G12V; (d) theDMS 53 cell line is modified in vitro to: 1) knockout expression ofCD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 26); and2) express GM-CSF (SEQ ID NO: 8), TGFI2 shRNA (SEQ ID NO: 24), IL-12(SEQ ID NO: 10), membrane bound CD40L (SEQ ID NO: 3), and TGFI1 shRNA(SEQ ID NO: 25); (e) the LK-2 cell line is modified in vitro to: 1)knockout expression of CD276 using a zinc-finger nuclease targetingCD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ ID NO: 8), membranebound CD40L (SEQ ID NO: 3), TGFI1 shRNA (SEQ ID NO: 25), and TGFI2 shRNA(SEQ ID NO: 24); and (f) the NCI-H23 cell line is modified in vitroto: 1) knockout expression of CD276 using a zinc-finger nucleasetargeting CD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ ID NO: 8),TGFI2 shRNA (SEQ ID NO: 24), IL-12 (SEQ ID NO: 10), membrane bound CD40L(SEQ ID NO: 3), TGFI1 shRNA (SEQ ID NO: 25), and modMSLN (SEQ ID NO:62).
 9. The kit of claim 8, wherein each vial comprises approximately1.0×10⁶-6.0×10⁷ cells.
 10. A unit dose comprising a first compositioncomprising cancer cell lines NCI-H460 (ATCC HTB-177), NCI-H520 (ATCCHTB-182) and A549 (ATCC CCL-185), and a second composition comprisingcancer cell lines DMS 53 (ATCC CRL-2062), NCI-H1703 (ATCC CRL-5889) andNCI-H23 (ATCC CRL-5800), wherein: (a) the NCI-H460 cell line is modifiedin vitro to: 1) knockout expression of CD276 using a zinc-fingernuclease targeting CD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ IDNO: 8), TGFβ2 shRNA (SEQ ID NO: 24), IL-12 (SEQ ID NO: 10), membranebound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 25), and modBORIS(SEQ ID NO: 60); (b) the NCI-H520 cell line is modified in vitro to: 1)knockout expression of CD276 using a zinc-finger nuclease targetingCD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ ID NO: 8), membranebound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 25), and TGFβ2 shRNA(SEQ ID NO: 24); (c) the A549 cell line is modified in vitro to: 1)knockout expression of CD276 using a zinc-finger nuclease targetingCD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ ID NO: 8), TGFβ2 shRNA(SEQ ID NO: 24), IL-12 (SEQ ID NO: 10), membrane bound CD40L (SEQ ID NO:3), TGFβ1 shRNA (SEQ ID NO: 25), modTBXT (SEQ ID NO: 79), modWT1 (SEQ IDNO: 81), and KRAS driver mutations G12D and G12V; (d) the DMS 53 cellline is modified in vitro to: 1) knockout expression of CD276 using azinc-finger nuclease targeting CD276 (SEQ ID NO: 26); and 2) expressGM-CSF (SEQ ID NO: 8), TGFβ2 shRNA (SEQ ID NO: 24), IL-12 (SEQ ID NO:10), membrane bound CD40L (SEQ ID NO: 3), and TGFβ1 shRNA (SEQ ID NO:25); (e) the NCI-H1703 cell line is modified in vitro to: 1) knockoutexpression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ IDNO: 26); and 2) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10),membrane bound CD40L (SEQ ID NO: 3), and TGFβ1 shRNA (SEQ ID NO: 25);and (f) the NCI-H23 cell line is modified in vitro to: 1) knockoutexpression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ IDNO: 26); and 2) express GM-CSF (SEQ ID NO: 8), TGFβ2 shRNA (SEQ ID NO:24), IL-12 (SEQ ID NO: 10), membrane bound CD40L (SEQ ID NO: 3), TGFβ1shRNA (SEQ ID NO: 25), and modMSLN (SEQ ID NO: 62).
 11. The unit dose ofclaim 10, wherein the first composition and second composition eachcomprise approximately 1.0×10⁶−6.0×10⁷ cells of each cell line.
 12. Theunit dose of claim 10, wherein the cell lines of (a)—(f) are irradiated.13. A composition comprising cancer cell lines DMS 53 (ATCC CRL-2062),NCI-H1703 (ATCC CRL-5889) and NCI-H23 (ATCC CRL-5800), wherein: (a) theDMS 53 cell line is modified in vitro to: 1) knockout expression ofCD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 26); and2) express GM-CSF (SEQ ID NO: 8), TGFβ2 shRNA (SEQ ID NO: 24), IL-12(SEQ ID NO: 10), membrane bound CD40L (SEQ ID NO: 3), and TGFβ1 shRNA(SEQ ID NO: 25); (b) the NCI-H1703 cell line is modified in vitro to: 1)knockout expression of CD276 using a zinc-finger nuclease targetingCD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQID NO: 10), membrane bound CD40L (SEQ ID NO: 3), and TGFβ1 shRNA (SEQ IDNO: 25); and (c) the NCI-H23 cell line is modified in vitro to: 1)knockout expression of CD276 using a zinc-finger nuclease targetingCD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ ID NO: 8), TGFβ2 shRNA(SEQ ID NO: 24), IL-12 (SEQ ID NO: 10), membrane bound CD40L (SEQ ID NO:3), TGFβ1 shRNA (SEQ ID NO: 25), and modMSLN (SEQ ID NO: 62).
 14. Thecomposition of claim 13, wherein the composition comprises approximately1.0×10⁷ cells for each cell line.
 15. A kit comprising six vials,wherein each vial individually comprises cells of cancer cell linesNCI-H460 (ATCC HTB-177), NCI-H520 (ATCC HTB-182) and A549 (ATCCCCL-185), DMS 53 (ATCC CRL-2062), NCI-H1703 (ATCC CRL-5889) and NCI-H23(ATCC CRL-5800), wherein: (a) the NCI-H460 cell line is modified invitro to: 1) knockout expression of CD276 using a zinc-finger nucleasetargeting CD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ ID NO: 8),TGFβ2 shRNA (SEQ ID NO: 24), IL-12 (SEQ ID NO: 10), membrane bound CD40L(SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 25), and modBORIS (SEQ ID NO:60); (b) the NCI-H520 cell line is modified in vitro to: 1) knockoutexpression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ IDNO: 26); and 2) express GM-CSF (SEQ ID NO: 8), membrane bound CD40L (SEQID NO: 3), TGFβ1 shRNA (SEQ ID NO: 25), and TGFβ2 shRNA (SEQ ID NO: 24);(c) the A549 cell line is modified in vitro to: 1) knockout expressionof CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 26);and 2) express GM-CSF (SEQ ID NO: 8), TGFβ2 shRNA (SEQ ID NO: 24), IL-12(SEQ ID NO: 10), membrane bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQID NO: 25), modTBXT (SEQ ID NO: 79), modWT1 (SEQ ID NO: 81), and KRASdriver mutations G12D and G12V; (d) the DMS 53 cell line is modified invitro to: 1) knockout expression of CD276 using a zinc-finger nucleasetargeting CD276 (SEQ ID NO: 26); and 2) express GM-CSF (SEQ ID NO: 8),TGFβ2 shRNA (SEQ ID NO: 24), IL-12 (SEQ ID NO: 10), membrane bound CD40L(SEQ ID NO: 3), and TGFβ1 shRNA (SEQ ID NO: 25); (e) the NCI-H1703 cellline is modified in vitro to: 1) knockout expression of CD276 using azinc-finger nuclease targeting CD276 (SEQ ID NO: 26); and 2) expressGM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane bound CD40L (SEQID NO: 3), and TGFβ1 shRNA (SEQ ID NO: 25); and (f) the NCI-H23 cellline is modified in vitro to: 1) knockout expression of CD276 using azinc-finger nuclease targeting CD276 (SEQ ID NO: 26); and 2) expressGM-CSF (SEQ ID NO: 8), TGFβ2 shRNA (SEQ ID NO: 24), IL-12 (SEQ ID NO:10), membrane bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 25),and modMSLN (SEQ ID NO: 62).
 16. The kit of claim 15, wherein each vialcomprises approximately 1.0×10⁶−6.0 ×10⁷ cells.